Antisense oligomer compounds

ABSTRACT

A modified antisense oligonucleotide of about 10 to about 40 nucleobases is disclosed. The oligonucleotide comprises a targeting sequence having a region complementary to at least one string of three or more identical contiguous nucleobases in a target sequence, wherein the target sequence comprises at least one additional nucleobase compared to the region of the targeting sequence and the at least one additional nucleobase has no complementary nucleobase in the region of the targeting sequence, and wherein the targeting region complementary to the at least one string of three or more identical contiguous nucleobases is internal to the targeting sequence.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/573,985 filed on Oct. 18, 2017 entitled ANTISENSE OLIGOMERCOMPOUNDS, the disclosure of which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

The present disclosure relates to an antisense oligomer compound inwhich a string of nucleobases present in the oligomer compound is brokenby a deletion of at least one nucleobase, and methods of using suchcompound.

BACKGROUND

Antisense oligomers offer great potential as pharmaceutical drugs, asevidenced by the number of antisense drugs currently in clinicaldevelopment, and aided by the fact that a number of potentiallimitations of antisense oligomers have been successfully addressed overthe past several years (see, for e.g.: Devi et al., 2002; AntisenseNucleic Acid Drug Dev.; see also, for e.g.: Stein et al., 2001;Antisense Nucleic Acid Drug Dev.). Novel uncharged oligomer backboneshave been developed to improve uptake into cells, and to increaseresistance to nuclease degradation (see, for e.g.: Iversen et al., 2001;Antisense Drug Technology). For some oligomer structures, for example,morpholino based structures, the modified backbone has been found togive enhanced binding affinity to its target nucleic acid (see, fore.g.: Iversen et al., 2001; Antisense Drug Technology; see also:Summerton et al., 1997; Antisense Nucleic Acid Drug Dev.).

In some antisense applications, the optimal targeting sequence againstwhich the antisense oligomer is directed to may include a biologicalpalindrome sequence or, alternately, a string of three or four or morenucleobases. Surprisingly, it has been found that antisense oligomercompounds directed towards a target sequence having a string of three ormore identical contiguous nucleobases, or alternatively a biologicalpalindrome sequence, can compromise the antisense activity of thecompound, as well as present difficulties in a manufacturing process.

SUMMARY

In various aspects, modifications to an antisense oligomer compound,having a string of three or four or more nucleobases, or alternatively,a biological palindrome sequence, is provided. In various embodiments,the modification enhances antisense activity of the compound and/or itsmethods of manufacture, including but not limited to aggregations thatcan occur during certain methods of manufacture.

In further aspects, a modified antisense oligonucleotide of about 10 toabout 40 nucleobases is provided. The subject oligonucleotide comprisesa targeting sequence having a region complementary to at least onestring of three or more identical contiguous nucleobases in a targetsequence, wherein the target sequence comprises at least one additionalnucleobase compared to the region of the targeting sequence and the atleast one additional nucleobase has no complementary nucleobase in theregion of the targeting sequence, and wherein the targeting regioncomplementary to the at least one string of three or more identicalcontiguous nucleobases is internal to the targeting sequence. Inembodiments, the targeting sequence comprises at least one string ofthree nucleobases. In embodiments, the targeting sequence comprises atleast one string of four nucleobases. In embodiments, the at least onestring of three or more nucleobases comprises at least one string ofthree or more guanine bases. In embodiments, the modified antisenseoligonucleotide is conjugated to a peptide. In embodiments, the targetsequence comprises an exon target associated with Duchenne musculardystrophy (DMD). In embodiments, the target sequence comprises exon 44in the processing of human dystrophin pre-processed mRNA. Inembodiments, the targeting sequence comprises any one of SEQ ID NOs:2-7. In embodiments, the target sequence comprises exons 45, 51 or 53 ofhuman dystrophin pre-processed mRNA. In embodiments, the targetingsequence comprises any one of SEQ ID NOs: 72-120. In embodiments, thetarget sequence comprises an exon target associated with spinal muscularatrophy (SMA). In embodiments, the target sequence comprises a regionadjacent to exon 7 in the processing of human SMN2 pre-processed mRNA.In embodiments, the targeting sequence comprises any one of SEQ ID NOs:9-25. In embodiments, the target sequence comprises an exon targetassociated with glycogen storage disease type 11 (GSD-II). Inembodiments, the target sequence comprises a region associated with exon2 of the human acid alpha-glucosidase pre-processed mRNA. Inembodiments, the targeting sequence comprises any one of SEQ ID NOs:26-68

In further aspects, the subject nucleotide comprises a targetingsequence having a region complementary to at least one biologicalpalindrome sequence in a target sequence, wherein the target sequencecomprises at least one additional nucleobase has no complementarynucleobase in the region of the targeting sequence, and wherein thetargeting region complementary to the at least one biological palindromesequence is internal to the targeting sequence. In embodiments, the atleast one biological palindrome sequence comprises at least five, atleast six, or at least seven or more nucleobases. In embodiments, themodified antisense oligonucleotide is conjugated to a peptide. Inembodiments, the target sequence comprises an exon target associatedwith Duchenne muscular dystrophy. In embodiments, the target sequencecomprises exon 44 in the processing of human dystrophin pre-processedmRNA. In embodiments, the targeting sequence comprises any one of SEQ IDNOs: 2-7. In embodiments, the target sequence comprises exons 45, 51 or53 of human dystrophin pre-processed mRNA. In embodiments, the targetingsequence comprises any one of SEQ ID NOs: 72-120. In embodiments, thetarget sequence comprises an exon target associated with spinal muscularatrophy. In embodiments, the target sequence comprises a region adjacentto exon 7 in the processing of human SMN2 pre-processed mRNA. Inembodiments, the targeting sequence comprises any one of SEQ ID NOs:9-25. In embodiments, the target sequence comprises an exon targetassociated with glycogen storage disease type II. In embodiments, thetarget sequence comprises a region associated with exon 2 of the humanacid alpha-glucosidase pre-processed mRNA. In embodiments, the targetingsequence comprises any one of SEQ ID NOs: 26-68.

In various aspects and embodiments, the subject oligonucleotide isreferred to as a deletion sequence. The deletion sequence comprises anyone of SEQ ID NOs: 1-128, wherein at least one nucleobase in any one ofSEQ ID NOs: 1-128 has been deleted. In embodiments, the at least onenucleobase that has been deleted is internal to the sequence of any oneof SEQ ID NOs: 1-128. In various embodiments, the deletion sequencecomprises CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 69; Eteplirsen);GTTGCCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 70; Golodirsen); orCAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 71; Casimersen). In variousembodiments, the deletion sequence comprises any one of SEQ ID NOs:69-71, wherein at least one nucleobase in any one of SEQ ID NOs: 69-71has been deleted. In embodiments, the at least one nucleobase that hasbeen deleted is internal to the sequence of any one of SEQ ID NOs:69-71.

These and other objects and features of the present disclosure willbecome more fully apparent when the following detailed description isread in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 details exon skipping percentages for deletion sequences directedagainst exon 44.

FIG. 2 details certain deletion sequences.

FIG. 3 details exon skipping percentages for deletion sequences detailedin FIG. 2 against a region adjacent to exons 7 and 8.

FIGS. 4 and 5 detail full-dose response experiments that identify asubset of 1-base-deletion sequences with high activity.

FIGS. 6A and 6B details SCX HPLC chromatograms (PMO and PPMO) for SEQ IDNO: 125. (A) details a chromatogram for SEQ ID NO: 125 PMO. (B) detailsa chromatogram for SEQ ID NO: 125 as a PPMO, showing unconjugated PMO,the expected main peak of conjugated PPMO, and higher molecular weightaggregates with the SEQ ID NO: 125 PPMO.

FIGS. 7A and 7B details SCX HPLC chromatograms (PMO and PPMO) for SEQ IDNO: 126. (A) details a chromatogram for SEQ ID NO: 126 PMO. (B) detailsa chromatogram for SEQ ID NO: 126 as a PPMO, showing unconjugated PMO,the expected main peak of conjugated PPMO, and, showing higher molecularweight aggregates with the SEQ ID NO: 126 PPMO.

DETAILED DESCRIPTION I. Definitions and Interpretation

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by those of ordinary skillin the art to which the disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the subject matter of the present disclosure,preferred methods and materials are described. For the purposes of thepresent disclosure, the following terms are defined below.

As used herein, the articles “a” and “an” are used herein to refer toone or to more than one (i.e., to at least one) of the grammaticalobject of the article. By way of example, “an element” means one elementor more than one element.

As used herein, “about” is meant a quantity, level, value, number,frequency, percentage, dimension, size, amount, weight or length thatvaries by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% toa reference quantity, level, value, number, frequency, percentage,dimension, size, amount, weight or length.

As used herein, unless the context requires otherwise, the words“comprise,” “comprises,” and “comprising” will be understood to implythe inclusion of a stated step or element or group of steps or elementsbut not the exclusion of any other step or element or group of steps orelements.

As used herein, “consisting of” is meant including, and limited to,whatever follows the phrase “consisting of:” Thus, the phrase“consisting of” indicates that the listed elements are required ormandatory, and that no other elements may be present. By “consistingessentially of” is meant including any elements listed after the phrase,and limited to other elements that do not interfere with or contributeto the activity or action specified in the disclosure for the listedelements. Thus, the phrase “consisting essentially of” indicates thatthe listed elements are required or mandatory, but that other elementsare optional and may or may not be present depending upon whether or notthey materially affect the activity or action of the listed elements.

As used herein, an “antisense oligonucleotide,” “antisense oligomer” or“oligonucleotide” refers to a linear sequence of nucleotides, ornucleotide analogs, which allows the nucleobase to hybridize to a targetsequence in an RNA by Watson-Crick base pairing, to form an oligomer RNAheteroduplex within the target sequence. The terms “antisenseoligonucleotide”, “modified antisense oligonucleotide”, “antisenseoligomer”, “oligomer” and “compound” may be used interchangeably torefer to an oligomer. The cyclic subunits may be based on ribose oranother pentose sugar or, in certain embodiments, a morpholino group(see description of morpholino oligomers herein). Also contemplated arepeptide nucleic acids (PNAs), locked nucleic acids (LNAs), tricyclo-DNAoligomers, tricyclo-phosphorothioate oligomers, and 2′-O-Methyloligomers, among other antisense agents known in the art. Included arenon-naturally-occurring oligomers, or “oligonucleotide analogs,”including oligomers having (i) a modified backbone structure, e.g., abackbone other than the standard phosphodiester linkage found innaturally-occurring oligo- and polynucleotides, and/or (ii) modifiedsugar moieties, e.g., morpholino moieties rather than ribose ordeoxyribose moieties. Oligomer analogs support bases capable of hydrogenbonding by Watson-Crick base pairing to standard polynucleotide bases,where the analog backbone presents the bases in a manner to permit suchhydrogen bonding in a sequence-specific fashion between the oligomeranalog molecule and bases in a standard polynucleotide (e.g.,single-stranded RNA or single-stranded DNA). Preferred analogs are thosehaving a substantially uncharged, phosphorus containing backbone.

As used herein, a “nuclease-resistant” oligomer refers to one whosebackbone is substantially resistant to nuclease cleavage, innon-hybridized or hybridized form; by common extracellular andintracellular nucleases in the body (for example, by exonucleases suchas 3′-exonucleases, endonucleases, RNase H); that is, the oligomer showslittle or no nuclease cleavage under normal nuclease conditions in thebody to which the oligomer is exposed. A “nuclease-resistantheteroduplex” refers to a heteroduplex formed by the binding of anantisense oligomer to its complementary target, such that theheteroduplex is substantially resistant to in vivo degradation byintracellular and extracellular nucleases, which are capable of cuttingdouble-stranded RNA/RNA or RNA/DNA complexes. A “heteroduplex” refers toa duplex between an antisense oligomer and the complementary portion ofa target RNA.

As used herein, “coding sequence” means any nucleic acid sequence thatcontributes to the code for the polypeptide product of a gene. Bycontrast, the term “non-coding sequence” refers to any nucleic acidsequence that does not directly contribute to the code for thepolypeptide product of a gene.

As used herein, the terms “contacting a cell”, “introducing” or“delivering” include delivery of the oligomers of the disclosure into acell by methods routine in the art, e.g., transfection (e.g., liposome,calcium-phosphate, polyethyleneimine), electroporation (e.g.,nucleofection), microinjection.

As used herein, the term “alkyl” is intended to include linear (i.e.,unbranched or acyclic), branched, cyclic, or polycyclic non-aromatichydrocarbon groups, which are optionally substituted with one or morefunctional groups. Unless otherwise specified, “alkyl” groups containone to eight, and preferably one to six carbon atoms. C₁-C₆ alkyl, isintended to include C1, C2, C3, C4, C5, and C6 alkyl groups. Lower alkylrefers to alkyl groups containing 1 to 6 carbon atoms. Examples of Alkylinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, pentyl,isopentyl tert-pentyl, cyclopentyl, hexyl, isohexyl, cyclohexyl, etc.Alkyl may be substituted or unsubstituted. Illustrative substitutedalkyl groups include, but are not limited to, fluoromethyl,difluoromethyl, trifluoromethyl, 2-fluoroethyl, 3-fluoropropyl,hydroxymethyl, 2-hydroxyethyl, 3-hydroxypropyl, benzyl, substitutedbenzyl, phenethyl, substituted phenethyl, etc.

As used herein, “alkenyl” refers to an unsaturated monovalent radicalcontaining carbon and hydrogen, which may be branched, linear, orcyclic. The alkenyl group may be monounsaturated or polyunsaturated.Generally preferred are alkenyl groups having one to six carbon atoms,referred to as “lower alkenyl”.

As used herein, the term “alkoxy” means a subset of alkyl in which analkyl group as defined above with the indicated number of carbonsattached through an oxygen bridge. For example, “alkoxy” refers togroups —O-alkyl, wherein the alkyl group contains 1 to 8 carbons atomsof a linear, branched, cyclic configuration. Examples of “alkoxy”include, but are not limited to, methoxy, ethoxy, n-propoxy, i-propoxy,t-butoxy, n-butoxy, s-pentoxy and the like.

As used herein, the term “aryl” used alone or as part of a larger moietyas in “aralkyl”, “aralkoxy”, or “aryloxy-alkyl”, refers to aromatic ringgroups having six to fourteen ring atoms, such as phenyl, 1-naphthyl,2-naphthyl, 1-anthracyl and 2-anthracyl. An “aryl” ring may contain oneor more substituents. The term “aryl” may be used interchangeably withthe term “aryl ring”. “Aryl” also includes fused polycyclic aromaticring systems in which an aromatic ring is fused to one or more rings.Non-limiting examples of useful aryl ring groups include phenyl,hydroxyphenyl, halophenyl, alkoxyphenyl, dialkoxyphenyl,trialkoxyphenyl, alkylenedioxyphenyl, naphthyl, phenanthryl, anthryl,phenanthro and the like, as well as 1-naphthyl, 2-naphthyl, 1-anthracyland 2-anthracyl. Also included within the scope of the term “aryl”, asit is used herein, is a group in which an aromatic ring is fused to oneor more non-aromatic rings, such as in a indanyl, phenanthridinyl, ortetrahydronaphthyl, where the radical or point of attachment is on thearomatic ring. “Aralkyl” refers to an alkyl, preferably lower (C₁-C₄,more preferably C₁-C₂) alkyl, substituent which is further substitutedwith an aryl group; examples are benzyl (—CH2C6H5) and phenethyl(—CH2CH2C6H5).

As used herein, the term “substituted”, with respect to an alkyl,alkenyl, alkynyl, aryl, aralkyl, or alkaryl group, refers to replacementof a hydrogen atom with a heteroatom-containing substituent, such as,for example, halogen, hydroxy, alkoxy, thiol, alkylthio, amino,alkylamino, imino, oxo (keto), nitro, cyano, or various acids or esterssuch as carboxylic, sulfonic, or phosphonic.

As used herein, the term “acyl” means a C(O)R group (in which Rsignifies H, alkyl or aryl as defined above). Examples of acyl groupsinclude formyl, acetyl, benzoyl, phenylacetyl and similar groups.

As used herein, the term “homolog” means compounds differing regularlyby the successive addition of the same chemical group. For example, ahomolog of a compound may differ by the addition of one or more —CH2-groups, amino acid residues, nucleotides, or nucleotide analogs.

As used herein, the terms “cell penetrating peptide” (CPP) or “a peptidemoiety which enhances cellular uptake” are used interchangeably andrefer to cationic cell penetrating peptides, also called “transportpeptides”, “carrier peptides”, or “peptide transduction domains”. Thepeptides, as shown herein, have the capability of inducing cellpenetration within about or at least about 30%, 40%, 50%, 60%, 70%, 80%,90%, or 100% of cells of a given cell culture population and allowmacromolecular translocation within multiple tissues in vivo uponsystemic administration. In some embodiments, the CPPs are of theformula —[(C(O)CHR′NH)m]R″ wherein R′ is a side chain of a naturallyoccurring amino acid or a one- or two-carbon homolog thereof, R″ isselected from hydrogen or acyl, and m is an integer up to 50. CPPs mayalso have the formula —[(C(O)CHR′NH)m]Ra wherein R′ is a side chain of anaturally occurring amino acid or a one- or two-carbon homolog thereof,and where Ra is selected from Hydrogen, acyl, benzoyl, or stearoyl. CPPsof any structure may be linked to the 3′ or 5′ end of an antisenseoligomer via a “linker” such as, for example, —C(O)(CH2)5NH—,—C(O)(CH2)2NH—, —C(O)(CH2)2NH—C(O)(CH2)5NH—, or —C(O)CH2NH—. AdditionalCPPs are well-known in the art and are disclosed, for example, in U.S.Patent Publication No. 2010/0016215, which is incorporated by referencein its entirety. In other embodiments, m is an integer selected from 1to 50 where, when m is 1, the moiety is a single amino acid orderivative thereof.

As used herein, “amino acid” refers to a compound consisting of a carbonatom to which are attached a primary amino group, a carboxylic acidgroup, a side chain, and a hydrogen atom. For example, the term “aminoacid” includes, but is not limited to, Glycine, Alanine, Valine,Leucine, Isoleucine, Asparagine, Glutamine, Lysine and Arginine.Additionally, as used herein, “amino acid” also includes derivatives ofamino acids such as esters, and amides, and salts, as well as otherderivatives, including derivatives having pharmacoproperties uponmetabolism to an active form. Accordingly, the term “amino acid” isunderstood to include naturally occurring and non-naturally occurringamino acids.

As used herein, an “electron pair” refers to a valence pair of electronsthat are not bonded or shared with other atoms.

As used herein, “homology” refers to the percentage number of aminoacids that are identical or constitute conservative substitutions.Homology may be determined using sequence comparison programs such asGAP (see: Deveraux et al., 1984, Nucleic Acids Research 12, 387-395). Inthis way sequences of a similar or substantially different length tothose cited herein could be compared by insertion of gaps into thealignment, such gaps being determined, for example, by the comparisonalgorithm used by GAP.

As used herein, “isolated” means material that is substantially oressentially free from components that normally accompany it in itsnative state. For example, an “isolated polynucleotide,” “isolatedoligonucleotide,” or “isolated oligomer” as used herein, may refer to apolynucleotide that has been purified or removed from the sequences thatflank it in a naturally-occurring state, e.g., a DNA fragment that isremoved from the sequences that are adjacent to the fragment in thegenome. The term “isolating” as it relates to cells refers to thepurification of cells (e.g., fibroblasts, lymphoblasts) from a sourcesubject (e.g., a subject with a polynucleotide repeat disease). In thecontext of mRNA or protein, “isolating” refers to the recovery of mRNAor protein from a source, e.g., cells.

As used herein, the terms “modulate” includes to “increase” or“decrease” one or more quantifiable parameters, optionally by a definedand/or statistically significant amount. By “increase” or “increasing,”“enhance” or “enhancing,” or “stimulate” or “stimulating,” refersgenerally to the ability of one or more antisense compounds orcompositions to produce or cause a greater physiological response (i.e.,downstream effects) in a cell or a subject relative to the responsecaused by either no antisense compound or a control compound. Relevantphysiological or cellular responses (in vivo or in vitro) will beapparent to persons skilled in the art. An “increased” or “enhanced”amount is typically a “statistically significant” amount, and mayinclude an increase that is 1.1, 1.2, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50 or more times (e.g., 500, 1000 times), including allintegers and decimal points in between and above 1 (e.g., 1.5, 1.6, 1.7.1.8), the amount produced by no antisense compound (the absence of anagent) or a control compound. The term “reduce” or “inhibit” may relategenerally to the ability of one or more antisense compounds orcompositions to “decrease” a relevant physiological or cellularresponse, such as a symptom of a disease or condition described herein,as measured according to routine techniques in the diagnostic art.Relevant physiological or cellular responses (in vivo or in vitro) willbe apparent to persons skilled in the art, and may include reductions inthe symptoms or pathology of a glycogen storage disease such as Pompedisease, for example, a decrease in the accumulation of glycogen in oneor more tissues. A “decrease” in a response may be “statisticallysignificant” as compared to the response produced by no antisensecompound or a control composition, and may include a 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 100% decrease, including all integers in between.

As used herein, “nucleobase” (Nu), “base pairing moiety” or “base” areused interchangeably to refer to a purine or pyrimidine base found innative DNA or RNA (uracil, thymine, adenine, cytosine, and guanine), aswell as analogs of the naturally occurring purines and pyrimidines, thatconfer improved properties, such as binding affinity to the oligomer.Exemplary analogs include hypoxanthine (the base component of thenucleoside inosine); 2, 6-diaminopurine; 5-methyl cytosine;C5-propynyl-modified pyrimidines; 9-(aminoethoxy)phenoxazine (G-clamp)and the like. Further examples of base pairing moieties include, but arenot limited to, uracil, thymine, adenine, cytosine, guanine andhypoxanthine having their respective amino groups protected by acylprotecting groups, 2-fluorouracil, 2-fluorocytosine, 5-bromouracil,5-iodouracil, 2,6-diaminopurine, azacytosine, pyrimidine analogs such aspseudoisocytosine and pseudouracil and other modified nucleobases suchas 8-substituted purines, xanthine, or hypoxanthine (the latter twobeing the natural degradation products). The modified nucleobasesdisclosed in Chiu and Rana, R N A, 2003, 9, 1034-1048, Limbach et al.Nucleic Acids Research, 1994, 22, 2183-2196 and Revankar and Rao,Comprehensive Natural Products Chemistry, vol. 7, 313, are alsocontemplated. Further examples of base pairing moieties include, but arenot limited to, expanded-size nucleobases in which one or more benzenerings has been added. Nucleic base replacements described in the GlenResearch catalog (www.glenresearch.com); Krueger A T et al, Acc. Chem.Res., 2007, 40, 141-150; Kool, ET, Acc. Chem. Res., 2002, 35, 936-943;Benner S. A., et al., Nat. Rev. Genet., 2005, 6, 553-543; Romesberg, F.E., et al., Curr. Opin. Chem. Biol., 2003, 7, 723-733; Hirao, I., Curr.Opin. Chem. Biol., 2006, 10, 622-627, are contemplated as useful for thesynthesis of the oligomers described herein. Examples of expanded-sizenucleobases are shown below:

A nucleobase covalently linked to a ribose, sugar analog or morpholinocomprises a nucleoside. “Nucleotides” are composed of a nucleosidetogether with one phosphate group. The phosphate groups covalently linkadjacent nucleotides to one another to form an oligomer.

As used herein, any of the terms “deletion sequence”, “gapmer”, or“blebmer” generally refer to an oligomer sequence of nucleobases thathas at least one fewer nucleobase compared to its target sequence. Invarious aspects and embodiments, any of the terms “deletion sequence”,“gapmer”, or “blebmer” specifically refer to a targeting sequence havinga region complementary to at least one string of three or more identicalcontiguous nucleobases in a target sequence, wherein the target sequencecomprises at least one additional nucleobase compared to the region ofthe targeting sequence and the at least one additional nucleobase has nocomplementary nucleobase in the region of the targeting sequence, andwherein the targeting region complementary to the at least one string ofthree or more identical contiguous nucleobases is internal to thetargeting sequence. In the alternative or in addition to the preceding,in further aspects and embodiments, any of the terms “deletionsequence”, “gapmer”, or “blebmer” can also specifically refer to atargeting sequence having a region complementary to at least onebiological palindrome sequence in a target sequence, wherein the targetsequence comprises at least one additional nucleobase has nocomplementary nucleobase in the region of the targeting sequence, andwherein the targeting region complementary to the at least onebiological palindrome sequence is internal to the targeting sequence.

As used herein, the term “biological palindrome sequence” refers to anoligonucleotide sequence wherein a portion of the oligonucleotidesequence, when read in reverse, is anti-sense to another portion of theoligonucleotide sequence.

As used herein, an oligomer “specifically hybridizes” to a targetpolynucleotide if the oligomer hybridizes to the target underphysiological conditions, with a Tm substantially greater than 40° C. or45° C., preferably at least 50° C., and typically 60° C.-80° C. orhigher. Such hybridization preferably corresponds to stringenthybridization conditions. At a given ionic strength and pH, the Tm isthe temperature at which 50% of a target sequence hybridizes to acomplementary polynucleotide. Such hybridization may occur with “near”or “substantial” complementarity of the antisense oligomer to the targetsequence, as well as with exact complementarity.

As used herein, “sufficient length” refers to an antisense oligomer or atargeting sequence thereof that is complementary to at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, or at least 30 or more,such as 8-40, and such as 15-40 contiguous nucleobases in a region, forexample, of GAA intron 1, exon 2, or intron 2, or a region spanning anyof the foregoing. An antisense oligomer of sufficient length has atleast a minimal number of nucleotides to be capable of specificallyhybridizing to a region, for example, of the GAA pre-mRNA repeat in themutant RNA. Preferably an oligomer of sufficient length is from 8 to 30nucleotides in length. More preferably, an oligomer of sufficient lengthis from 9 to 27 nucleotides in length. Even more preferably, an oligomerof sufficient length is from 15 to 40 nucleotides in length.

As used herein, the terms “sequence identity” or, for example,comprising a “sequence 50% identical to,” refer to the extent thatsequences are identical on a nucleotide-by-nucleotide basis or an aminoacid-by-amino acid basis over a window of comparison. Thus, a“percentage of sequence identity” may be calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, I) or the identical amino acid residue (e.g., Ala, Pro, Ser,Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn,Gln, Cys and Met) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. Optimal alignment of sequences for aligning a comparisonwindow may be conducted by computerized implementations of algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Drive Madison,Wis., USA) or by inspection and the best alignment (i.e., resulting inthe highest percentage homology over the comparison window) generated byany of the various methods selected. Reference also may be made to theBLAST family of programs as for example disclosed by Altschul et al.,Nucl. Acids Res. 25:3389, 1997.

As used herein, a “subject” or a “subject in need thereof” includes, butis not limited to, a mammalian subject such as a human subject.Exemplary mammalian subjects have or are at risk for having GSD-II (orPompe disease), or SMA, or DMD.

As used herein, the term “target” refers to a RNA region, and as anon-limiting example, to a region identified by the GAA gene. In anon-limiting embodiment the target is a region within intron 1 of theGAA-coding pre-mRNA, which is responsible for suppression of a signalthat promotes exon 2 inclusion. In another embodiment the target regionis a region of the mRNA of GAA exon 2. In a further embodiment, thetarget comprises one or more discrete subregions of intron 1 of theGAA-coding pre-mRNA.

As used herein, the term “target sequence” refers to a portion of thetarget RNA against which the oligomer analog is directed, that is, thesequence to which the oligomer analog will hybridize by Watson-Crickbase pairing of a complementary sequence.

As used herein, the term “targeting sequence” is the sequence in theoligomer or oligomer analog that is complementary (meaning, in addition,substantially complementary) to the “target sequence” in the RNA genome.The entire sequence, or only a portion, of the antisense oligomer may becomplementary to the target sequence. For example, in an oligomer having20-30 bases, about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 may be targeting sequencesthat are complementary to the target region. Typically, the targetingsequence is formed of contiguous bases in the oligomer, but mayalternatively be formed of non-contiguous sequences that when placedtogether, e.g., from opposite ends of the oligomer, constitute sequencethat spans the target sequence.

As used herein, “targeting sequence” may have “near” or “substantial”complementarity to the target sequence and still function for thepurpose of the present disclosure, that is, still be “complementary.” Inembodiments, the oligomer analog compounds employed in the presentdisclosure have at most one mismatch with the target sequence out of 10nucleotides, and preferably at most one mismatch out of 20. Inembodiments, the oligomer analog compounds employed in the presentdisclosure have at least one mismatch with the target sequence out of 10nucleotides, and preferably at least one mismatch out of 20.Alternatively, the antisense oligomers employed have at least 90%sequence homology, and preferably at least 95% sequence homology, withthe exemplary targeting sequences as designated herein.

As used herein, the terms “TEG” or “triethylene glycol tail” refer totriethylene glycol moieties conjugated to the oligonucleotide, e.g., atits 3′- or 5′-end. For example, in some embodiments, “TEG” includeswherein, for example, T of the compound of formula (I), (VI), or (VII)is of the formula:

As used herein, the term “quantifying”, “quantification” or otherrelated words refer to determining the quantity, mass, or concentrationin a unit volume, of a nucleic acid, polynucleotide, oligomer, peptide,polypeptide, or protein.

As used herein, “treatment” of a subject (e.g., a mammal, such as ahuman) or a cell is any type of intervention used in an attempt to alterthe natural course of the individual or cell. Treatment includes, but isnot limited to, administration of a pharmaceutical composition, and maybe performed either prophylactically or subsequent to the initiation ofa pathologic event or contact with an etiologic agent. Also included are“prophylactic” treatments, which can be directed to reducing the rate ofprogression of the disease or condition being treated, delaying theonset of that disease or condition, or reducing the severity of itsonset. “Treatment” or “prophylaxis” does not necessarily indicatecomplete eradication, cure, or prevention of the disease or condition,or associated symptoms thereof.

As used herein, a “heterocycle” refers to a non-aromatic ring,preferably a 5- to 7-membered ring, whose ring atoms are selected fromthe group consisting of carbon, nitrogen, oxygen and sulfur. Preferably,the ring atoms include 3 to 6 carbon atoms. Such heterocycles include,for example, pyrrolidine, piperidine, piperazine, and morpholine.

As used herein, a “morpholino oligomer” is an oligonucleotide analogcomposed of morpholino subunit structures, where (i) the structures arelinked together by phosphorus-containing linkages, one to three atomslong, preferably two atoms long, and preferably uncharged, joining themorpholino nitrogen of one subunit to the 5′ exocyclic carbon of anadjacent subunit, and (ii) Pi and Pj are purine or pyrimidinebase-pairing moieties effective to bind, by base-specific hydrogenbonding, to a base in a polynucleotide. The purine or pyrimidinebase-pairing moiety is typically adenine, cytosine, guanine, uracil orthymine. The synthesis, structures, and binding characteristics ofmorpholino oligomers are detailed in U.S. Pat. Nos. 5,698,685;5,217,866; 5,142,047; 5,034,506; 5,166,315; 5,521,063; and 5,506,337,all of which are incorporated herein by reference. Desirable chemicalproperties of the morpholino-based oligomers include the ability toselectively hybridize with a complementary-base target nucleic acid,including target RNA, with high Tm, even with oligomers as short as 8-14bases, the ability to be actively transported into mammalian cells, andthe ability of an oligomer RNA heteroduplex to resist RNAse degradation.

As used herein, a “substantially uncharged” morpholino oligomer includesat most one charged intersubunit linkage for every four, preferably forevery ten, and more preferably for every twenty, uncharged intersubunitlinkages. Any charged linkages are preferably charged phosphoramidate(or thiophosphoramidate) linkages. Preferably, the morpholino oligomersare fully uncharged.

As used herein, an “amino acid subunit” is preferably an α-amino acidresidue (i.e. —CO—CHR—NH—); it may also be a β- or other amino acidresidue (e.g. —CO—CH2CHR—NH—), where R is a side chain.

As used herein, a “G-quartet” includes a stacked planar hydrogen-bondedguanine tetramers that can cause guanine-rich nucleic acids to adoptintermolecular and intramolecular quadruplex structures that arestabilized by the presence of the G-quartets.

As used herein, the term “non-natural amino acids” refers to those aminoacids not present in proteins found in nature such as beta-alanine(H-Ala) or 6-aminohexanoic acid (Ahx).

As used herein, the abbreviation “DMD” refers to Duchenne musculardystrophy.

As used herein, the abbreviation “SMA” refers to spinal muscularatrophy.

II. Description of the Disclosure A. Antisense Oligomer Compounds

In an aspect, the antisense oligomer compound is a synthetic oligomercapable of base-specific binding to a target sequence of apolynucleotide, e.g., an antisense oligonucleotide analog. Such analogs,in which the backbone structure, ring structure, or, less frequently,base structure of natural polynucleotides is modified, are well knownand include charged analogs, such as phosphorothioate-linkedoligonucleotides, and uncharged analogs, such as methylphosphonates andpeptide nucleic acids. Some analogs, such as N3′→P5′ phosphoramidates,may be charged or uncharged, depending on the substation on the linkingmoiety.

In embodiments, the antisense oligomer compound is a morpholinooligomer, as defined above, which is about 8-40 subunits in length. Moretypically, the oligomer is about 10-30, or about 12-25, subunits inlength. For some applications, such as antibacterial, short oligomers,e.g. from about 8-12 subunits in length, can be especially advantageous,particularly when attached to a peptide transporter as disclosed herein.Preferably, the oligomer is an uncharged phosphorodiamidate-linkedmorpholino oligomer (PMO), also defined above. The PMO can be of anysequence, where the supported base pairing groups include standard ormodified A, T, C, G, I and U bases.

In an aspect, the target nucleic acid sequence against which theoligomer compound is directed includes a region having a string of threeor more identical contiguous nucleobases. In embodiments, the three ormore identical contiguous nucleobases are three identical contiguousnucleobases. In embodiments, the three or more identical contiguousnucleobases are four identical contiguous nucleobases. In embodiments,the three or more identical contiguous nucleobases are five identicalcontiguous nucleobases. In embodiments, the three or more identicalcontiguous nucleobases are six identical contiguous nucleobases. Inembodiments, the three or more identical contiguous nucleobases areseven identical contiguous nucleobases. In embodiments, the three ormore identical contiguous nucleobases are eight identical contiguousnucleobases. In embodiments, the three or more identical contiguousnucleobases are nine or more identical contiguous nucleobases.

In embodiments, the three or more identical contiguous nucleobases arereduced to two identical contiguous nucleobases. In embodiments, thethree identical contiguous nucleobases are reduced to two identicalcontiguous nucleobases (i.e., one nucleobase is removed). Inembodiments, the four identical contiguous nucleobases are reduced totwo identical contiguous nucleobases (i.e., two nucleobases areremoved). In embodiments, the five identical contiguous nucleobases arereduced to two identical contiguous nucleobases (i.e., three nucleobasesare removed). In embodiments, the six identical contiguous nucleobasesare reduced to two identical contiguous nucleobases (i.e., fournucleobases are removed). The same nucleobase removal approach can bemade to more than six identical contiguous nucleobases.

In further embodiments, the string of three or more identical contiguousnucleobases is a string of three or more contiguous G nucleobases. Inembodiments, the string of three or more contiguous G nucleobases isthree contiguous G nucleobases. In embodiments, the string of three ormore contiguous G nucleobases is four, five, six, seven, eight, nine, ormore contiguous G nucleobases.

In embodiments, the three or more contiguous G nucleobases are reducedto two contiguous G nucleobases. In embodiments, three contiguous Gnucleobases are reduced to two contiguous G nucleobases (i.e., one Gnucleobase is removed). In embodiments, four contiguous G nucleobasesare reduced to two contiguous G nucleobases (i.e., two G nucleobases areremoved). In embodiments, five contiguous G nucleobases are reduced totwo contiguous G nucleobases (i.e., three G nucleobases are removed). Inembodiments, six contiguous G nucleobases are reduced to two contiguousG nucleobases (i.e., four G nucleobases are removed). The samenucleobase removal approach can be made to more than six identicalcontiguous G nucleobases.

According to an aspect, the target nucleic acid sequence against whichthe oligomer compound is directed includes a region having a biologicalpalindrome sequence. Alternatively, the target region may include or beadjacent to a donor or acceptor splice site in a preprocessed mRNA,where it is desired to block correct splicing at that site, either forpurposes of creating splice mutation polypeptides, or incomplete orinactive peptides. In still another embodiment, the target may be acis-acting element in a viral genome, where binding of the oligomer(which may be targeted against either the + or − viral genome strand),is effective to block viral replication in virus-infected cells.

Exemplary target sequences containing a string of three or four or morenucleobases or a biological palindrome sequence in each of these targettypes can be found from public sequence databases known to those ofskill in the art. It is to be appreciated, however, that variousdeletions in the targeting sequence described throughout this disclosureare illustrative of how the oligomer compound may be modified to achievethe advantages of the invention.

The transporter can be linked to the compound to be delivered by avariety of methods available to one of skill in the art. In one example,the transporter is a peptide containing a single cysteine residue whoseside chain thiol is used for linking. The linkage point can be atvarious locations along the transporter. In selected embodiments, it isat a terminus of the transporter. Typically, it is adjacent to thehydrophobic residues of the transporter. Multiple transporters can beattached to a single compound if desired.

The linker can also be any combination of two β-Ala and/or Ahx residuesattached to the 5′ end of the PMO and the C-terminus of the peptidetransporter. A preferred embodiment is to attach the Ahx residue to theC terminus of the peptide transporter and the β-Ala residue to the 5′terminus of the PMO.

When the compound is a PMO, the transporter can be attached at the 5′end of the PMO, e.g., via the 5′-hydroxyl group, or via an amine cappingmoiety. Alternatively, the transporter may be attached at the 3′ end,e.g. via a morpholino ring nitrogen, or via the side chain of anintersubunit linkage, either at a terminus or an internal linkage. Thelinker may also comprise a direct bond between the carboxy terminus of atransporter peptide and an amine or hydroxy group of the PMO, formed bycondensation promoted by e.g., carbodiimide.

Linkers can be selected from those which are non-cleavable under normalconditions of use, e.g., containing a thioether or carbamate bond. Insome embodiments, it may be desirable to include a linkage between thetransporter moiety and compound which is cleavable in vivo. Bonds whichare cleavable in vivo are known in the art and include, for example,carboxylic acid esters, which are hydrolyzed enzymatically, anddisulfides, which are cleaved in the presence of glutathione. It mayalso be feasible to cleave a photolytically cleavable linkage, such asan ortho-nitrophenyl ether, in vivo by application of radiation of theappropriate wavelength.

For example, the preparation of a conjugate having a disulfide linker,using the reagent N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP)or succinimidyloxycarbonyl α-methyl-α-(2-pyridyldithio) toluene (SMPT).Exemplary heterobifunctional linking agents which further contain acleavable disulfide group include N-hydroxysuccinimidyl3-[(4-azidophenyl)dithio]propionate and others described in (See: Vaninand Ji, 1981).

In embodiments, antisense targeting sequences are designed to hybridizeto a region of one or more of the target sequences. Selected antisensetargeting sequences can be made shorter, e.g., about 12 bases, orlonger, e.g., about 40 bases, and include a small number of mismatches,as long as the sequence is sufficiently complementary to effect splicemodulation upon hybridization to the target sequence, and optionallyforms with the RNA a heteroduplex having a Tm of 45° C. or greater.

In embodiments, the degree of complementarity between the targetsequence and antisense targeting sequence is sufficient to form a stableduplex. The region of complementarity of the antisense oligomers withthe target RNA sequence may be as short as 8-11 bases, but can be 12-15bases or more, e.g., 10-40 bases, 12-30 bases, 12-25 bases, 15-25 bases,12-20 bases, or 15-20 bases, including all integers in between theseranges. An antisense oligomer of about 14-15 bases is generally longenough to have a unique complementary sequence. In certain embodiments,a minimum length of complementary bases may be required to achieve therequisite binding Tm, as discussed herein.

In embodiments, oligomers as long as 40 bases may be suitable, where atleast a minimum number of bases, e.g., 10-12 bases, are complementary tothe target sequence. In some embodiments, facilitated or active uptakein cells is optimized at oligomer lengths of less than about 30 bases.For PMO oligomers, described further herein, an optimum balance ofbinding stability and uptake generally occurs at lengths of 18-25 bases.Included in the disclosure are antisense oligomers (e.g., PMOs, PMO-X,PNAs, LNAs, 2′-OMe) that consist of about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40 bases, in which at least about 6, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 contiguous ornon-contiguous bases are complementary to the target sequences.

In embodiments, the antisense oligomers typically comprise a basesequence which is sufficiently complementary to a sequence or regionwithin or adjacent to intron 1, exon 2, or intron 2 of the pre-mRNAsequence of the human GAA gene. Ideally, an antisense oligomer is ableto effectively modulate aberrant splicing of the GAA pre-mRNA, andthereby increase expression of active GAA protein. This requirement isoptionally met when the oligomer compound has the ability to be activelytaken up by mammalian cells, and once taken up, form a stable duplex (orheteroduplex) with the target mRNA, optionally with a Tm greater thanabout 40° C. or 45° C.

In embodiments, the antisense oligomers typically comprise a basesequence which is sufficiently complementary to a sequence or regionwithin or adjacent to exons associated with DMD, SMA, or Pompe disease.

In embodiments, antisense oligomers may be 100% complementary to thetarget sequence, or may include mismatches, e.g., to accommodatevariants, as long as a heteroduplex formed between the oligomer andtarget sequence is sufficiently stable to withstand the action ofcellular nucleases and other modes of degradation which may occur invivo. Hence, certain oligomers may have substantial complementarity,meaning, about or at least about 70% sequence complementarity, e.g.,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence complementarity, between the oligomer and thetarget sequence. Oligomer backbones that are less susceptible tocleavage by nucleases are discussed herein. Mismatches, if present, aretypically less destabilizing toward the end regions of the hybrid duplexthan in the middle. The number of mismatches allowed will depend on thelength of the oligomer, the percentage of G:C base pairs in the duplex,and the position of the mismatch(es) in the duplex, according to wellunderstood principles of duplex stability. Although such an antisenseoligomer is not necessarily 100% complementary to the v target sequence,it is effective to stably and specifically bind to the target sequence,such that splicing of the target pre-RNA is modulated.

The stability of the duplex formed between an oligomer and a targetsequence is a function of the binding Tm and the susceptibility of theduplex to cellular enzymatic cleavage. The Tm of an oligomer withrespect to complementary-sequence RNA may be measured by conventionalmethods, such as those described by Hames et al., Nucleic AcidHybridization, IRL Press, 1985, pp. 107-108 or as described in Miyada C.G. and Wallace R. B., 1987, Oligomer Hybridization Techniques, MethodsEnzymol. Vol. 154 pp. 94-107. In certain embodiments, antisenseoligomers may have a binding Tm, with respect to acomplementary-sequence RNA, of greater than body temperature andpreferably greater than about 45° C. or 50° C. Tm's in the range 60-80°C. or greater are also included. According to well-known principles, theTm of an oligomer, with respect to a complementary-based RNA hybrid, canbe increased by increasing the ratio of C:G paired bases in the duplex,and/or by increasing the length (in base pairs) of the heteroduplex. Atthe same time, for purposes of optimizing cellular uptake, it may beadvantageous to limit the size of the oligomer. For this reason,compounds that show high Tm (45-50° C. or greater) at a length of 25bases or less are generally preferred over those requiring greater than25 bases for high Tm values.

The activity of antisense oligomers and variants thereof can be assayedaccording to routine techniques in the art. For example, splice formsand expression levels of surveyed RNAs and proteins may be assessed byany of a wide variety of well-known methods for detecting splice formsand/or expression of a transcribed nucleic acid or protein. Non-limitingexamples of such methods include RT-PCR of spliced forms of RNA followedby size separation of PCR products, nucleic acid hybridization methodse.g., Northem blots and/or use of nucleic acid arrays; nucleic acidamplification methods; immunological methods for detection of proteins;protein purification methods; and protein function or activity assays.

RNA expression levels can be assessed by preparing mRNA/cDNA (i.e., atranscribed polynucleotide) from a cell, tissue or organism, and byhybridizing the mRNA/cDNA with a reference polynucleotide that is acomplement of the assayed nucleic acid, or a fragment thereof cDNA can,optionally, be amplified using any of a variety of polymerase chainreaction or in vitro transcription methods prior to hybridization withthe complementary polynucleotide; preferably, it is not amplified.Expression of one or more transcripts can also be detected usingquantitative PCR to assess the level of expression of the transcript(s).

B. Antisense Oligomer Chemistries

i. General Characteristics

Certain antisense oligomers of the present disclosure specificallyhybridize to an intronic splice silencer element or an exonic splicesilencer element. In certain embodiments, the antisense oligomercomprises a non-natural chemical backbone selected from aphosphoramidate or phosphorodiamidate morpholino oligomer (PMO), apeptide nucleic acid (PNA), a locked nucleic acid (LNA), aphosphorothioate oligomer, a tricyclo-DNA oligomer, atricyclo-phosphorothioate oligomer, a 2′O-Me-modified oligomer, or anycombination of the foregoing, and a targeting sequence complementary toa region within intron 1, intron 2, or exon 2 of a pre-mRNA of the humanacid alpha-glucosidase (GAA) gene.

Antisense oligomers of the present disclosure generally comprise aplurality of nucleotide subunits each bearing a nucleobase which takentogether form or comprise a targeting sequence. Accordingly, in someembodiments, the antisense oligomers range in length from about 10 toabout 40 subunits, more preferably about 10 to 30 subunits, andtypically 15-25 subunits. For example, antisense compounds of thedisclosure may be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or40 subunits in length, or range from 10 subunits to 40 subunits, 10subunits to 30 subunits, 14 subunits to 25 subunits, 15 subunits to 30subunits, 17 subunits to 30 subunits, 17 subunits to 27 subunits, 10subunits to 27 subunits, 10 subunits to 25 subunits, and 10 subunits to20 subunits. In certain embodiments, the antisense oligomer is about 10to about 40 or about 5 to about 30 nucleotides in length. In someembodiments, the antisense oligomer is about 14 to about 25 or about 17to about 27 nucleotides in length.

In various embodiments, an antisense oligomer comprises a completelymodified backbone, for example, 100% of the backbone is modified (forexample, a 25 mer antisense oligomer comprises its entire backbonemodified with any combination of the backbone modifications as describedherein). In various embodiments, an antisense oligomer may compriseabout 100% to 2.5% of its backbone modified. In various embodiments, anantisense oligomer may comprise about 99%, 95%, 90%, 85%, 80%, 75%, 70%,65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 2.5%of its backbone modified, and iterations in between. In otherembodiments, an antisense oligomer may comprise any combination ofbackbone modifications as described herein.

In various embodiments, an antisense oligomer comprises, consists of, orconsists essentially of phosphoramidate morpholino oligomers andphosphorodiamidate morpholino oligomers (PMO), phosphorothioate modifiedoligomers, 2′ O-methyl modified oligomers, peptide nucleic acid (PNA),locked nucleic acid (LNA), phosphorothioate oligomers, 2′ O-MOE modifiedoligomers, 2′-fluoro-modified oligomer, 2′O,4′C-ethylene-bridged nucleicacids (ENAs), tricyclo-DNAs, tricyclo-DNA phosphorothioate nucleotides,2′-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers, morpholinooligomers, peptide-conjugated phosphoramidate morpholino oligomers(PPMO), phosphorodiamidate morpholino oligomers having a phosphorousatom with (i) a covalent bond to the nitrogen atom of a morpholino ring,and (ii) a second covalent bond to a (1,4-piperazin)-1-yl substituent orto a substituted (1,4-piperazin)-1-yl (PMOplus), and phosphorodiamidatemorpholino oligomers having a phosphorus atom with (i) a covalent bondto the nitrogen atom of a morpholino ring and (ii) a second covalentbond to the ring nitrogen of a 4-aminopiperdin-1-yl (i.e., APN) or aderivative of 4-aminopiperdin-1-yl (PMO-X) chemistries, includingcombinations of any of the foregoing.

In an aspect, the antisense oligomers described herein comprise anantisense PPMO oligomer. In embodiments, the antisense PPMO oligomercomprises three, four, five, six, seven, eight, nine or more internalcontiguous nucleobases. In embodiments, removal of one or more of thecontiguous nucleobases is made such that there are only two contiguousnucleobases, which results in a removal of aggregated structures.

In embodiments, the antisense PPMO oligomer comprises three, four, five,six, seven, eight, nine or more internal contiguous G nucleobases. Inembodiments, removal of one or more of the contiguous G nucleobases ismade such that there are only two contiguous G nucleobases, whichresults in a removal of aggregated structures.

In various embodiments, the backbone of the antisense oligomer issubstantially uncharged, and is optionally recognized as a substrate foractive or facilitated transport across the cell membrane. In someembodiments, all the internucleoside linkages are uncharged. The abilityof the oligomer to form a stable duplex with the target RNA may alsorelate to other features of the backbone, including the length anddegree of complementarity of the antisense oligomer with respect to thetarget, the ratio of G:C to A:T base matches, and the positions of anymismatched bases. The ability of the antisense oligomer to resistcellular nucleases may promote survival and ultimate delivery of theagent to the cell cytoplasm.

In certain embodiments, the antisense oligomer has at least oneinternucleoside linkage that is positively charged or cationic atphysiological pH. In some embodiments, the antisense oligomer has atleast one internucleoside linkage that exhibits a pKa between about 5.5and about 12. In further embodiments, the antisense oligomer containsabout, at least about, or no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 internucleoside linkages that exhibits a pKa between about 4.5 andabout 12. In some embodiments, the antisense oligomer contains about orat least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% internucleoside linkages thatexhibit a pKa between about 4.5 and about 12. Optionally, the antisenseoligomer has at least one internucleoside linkage with both a basicnitrogen and an alkyl, aryl, or aralkyl group. In particularembodiments, the cationic interucleoside linkage or linkages comprise a4-aminopiperdin-1-yl (APN) group, or a derivative thereof. While notbeing bound by any one theory, it is believed that the presence of acationic linkage or linkages (e.g., APN group or APN derivative) in theoligomer facilitates binding to the negatively charged phosphates in thetarget nucleotide. Thus, the formation of a heteroduplex between mutantRNA and the cationic linkage-containing oligomer may be held together byboth an ionic attractive force and Watson-Crick base pairing.

In some embodiments, the number of cationic linkages is at least 2 andno more than about half the total internucleoside linkages, e.g., aboutor no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20 cationic linkages. In some embodiments, however, upto all of the internucleoside linkages are cationic linkages, e.g.,about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31. 32,33, 34, 35, 36, 37, 38, 39, or 40 of the total internucleoside linkagesare cationic linkages. In specific embodiments, an oligomer of about19-20 subunits may have 2-10, e.g., 4-8, cationic linkages, and theremainder uncharged linkages. In other specific embodiments, an oligomerof 14-15 subunits may have 2-7, e.g., 2, 3, 4, 5, 6, or 7 cationiclinkages and the remainder uncharged linkages. The total number ofcationic linkages in the oligomer can thus vary from about 1 to 10 to 15to 20 to 30 or more (including all integers in between), and can beinterspersed throughout the oligomer.

In various embodiments, an antisense oligomer may have about or up toabout 1 cationic linkage per every 2-5 or 2, 3, 4, or 5 unchargedlinkages, such as about 4-5 or 4 or 5 per every 10 uncharged linkages.

Certain embodiments include antisense oligomers that contain about 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% cationic linkages. In certain embodiments,optimal improvement in antisense activity may be seen if about 25% ofthe backbone linkages are cationic. In certain embodiments, enhancementmay be seen with a small number e.g., 10-20% cationic linkages, or wherethe number of cationic linkages is in the range 50-80%, such as about60%.

In various embodiments, the cationic linkages are interspersed along thebackbone. Such oligomers optionally contain at least two consecutiveuncharged linkages; that is, the oligomer optionally does not have astrictly alternating pattern along its entire length. In specificinstances, each one or two cationic linkage(s) is/are separated alongthe backbone by at least 1, 2, 3, 4, or 5 uncharged linkages.

Also included are oligomers having blocks of cationic linkages andblocks of uncharged linkages. For example, a central block of unchargedlinkages may be flanked by blocks of cationic linkages, or vice versa.In some embodiments, the oligomer has approximately equal-length 5′, 3′and center regions, and the percentage of cationic linkages in thecenter region is greater than about 50%, 60%, 70%, or 80% of the totalnumber of cationic linkages.

In certain antisense oligomers, the bulk of the cationic linkages (e.g.,70, 75%, 80%, 90% of the cationic linkages) are distributed close to the“center-region” backbone linkages, e.g., the 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 centermost linkages. For example, a 16, 17, 18, 19, 20, 21,22, 23, or 24-mer oligomer with may have at least 50%, 60%, 70%, or 80%of the total cationic linkages localized to the 8, 9, 10, 11, or 12centermost linkages.

ii. Backbone Chemistry Features

The antisense oligomers can employ a variety of antisense chemistries.Examples of oligomer chemistries include, without limitation,phosphoramidate morpholino oligomers and phosphorodiamidate morpholinooligomers (PMO), phosphorothioate modified oligomers, 2′ O-methylmodified oligomers, peptide nucleic acid (PNA), locked nucleic acid(LNA), phosphorothioate oligomers, 2′ O-MOE modified oligomers,2′-fluoro-modified oligomer, 2′O,4′C-ethylene-bridged nucleic acids(ENAs), tricyclo-DNAs, tricyclo-DNA phosphorothioate nucleotides,2′-O-[2-(N-methylcarbamoyl)ethyl] modified oligomers, morpholinooligomers, peptide-conjugated phosphoramidate morpholino oligomers(PPMO), phosphorodiamidate morpholino oligomers having a phosphorousatom with (i) a covalent bonds to the nitrogen atom of a morpholinoring, and (ii) a second covalent bond to a (1,4-piperazin)-1-ylsubstituent or to a substituted (1,4-piperazin)-1-yl (PMOplus), andphosphorodiamidate morpholino oligomers having a phosphorus atom with(i) a covalent bond to the nitrogen atom of a morpholino ring and (ii) asecond covalent bond to the ring nitrogen of a 4-aminopiperdin-1-yl(i.e., APN) or a derivative of 4-aminopiperdin-1-yl (PMO-X) chemistries,including combinations of any of the foregoing. In general, PNA and LNAchemistries can utilize shorter targeting sequences because of theirrelatively high target binding strength relative to PMO and 2′O-Memodified oligomers. Phosphorothioate and 2′O-Me-modified chemistries canbe combined to generate a 2′O-Me-phosphorothioate backbone. See, e.g.,PCT Publication Nos. WO/2013/112053 and WO/2009/008725, which are herebyincorporated by reference in their entireties.

In some instances, antisense oligomers such as PMOs can be conjugated tocell penetrating peptides (CPPs) to facilitate intracellular delivery.Peptide-conjugated PMOs are called PPMOs and certain embodiments includethose described in PCT Publication No. WO/2012/150960, incorporatedherein by reference in its entirety. In some embodiments, anarginine-rich peptide sequence conjugated or linked to, for example, the3′ terminal end of an antisense oligomer as described herein may beused. In certain embodiments, an arginine-rich peptide sequenceconjugated or linked to, for example, the 5′ terminal end of anantisense oligomer as described herein may be used.

1. Peptide Nucleic Acids (PNAs)

Peptide nucleic acids (PNAs) are analogs of DNA in which the backbone isstructurally homomorphous with a deoxyribose backbone, consisting ofN-(2-aminoethyl) glycine units to which pyrimidine or purine bases areattached. PNAs containing natural pyrimidine and purine bases hybridizeto complementary oligomers obeying Watson-Crick base-pairing rules, andmimic DNA in terms of base pair recognition (See: Egholm, Buchardt etal., 1993). The backbone of PNAs is formed by peptide bonds rather thanphosphodiester bonds, making them well-suited for antisense applications(see structure below). The backbone is uncharged, resulting in PNA/DNAor PNA/RNA duplexes that exhibit greater than normal thermal stability.PNAs are not recognized by nucleases or proteases. A non-limitingexample of a PNA is depicted below:

Despite a radical structural change to the natural structure, PNAs arecapable of sequence-specific binding in a helix form to DNA or RNA.Characteristics of PNAs include a high binding affinity to complementaryDNA or RNA, a destabilizing effect caused by single-base mismatch,resistance to nucleases and proteases, hybridization with DNA or RNAindependent of salt concentration and triplex formation with homopurineDNA. PANAGENE™ has developed its proprietary Bts PNA monomers (Bts;benzothiazole-2-sulfonyl group) and proprietary oligomerization process.The PNA oligomerization using Bts PNA monomers is composed of repetitivecycles of deprotection, coupling and capping. PNAs can be producedsynthetically using any technique known in the art. See, e.g., U.S. Pat.Nos. 6,969,766, 7,211,668, 7,022,851, 7,125,994, 7,145,006 and7,179,896. See also U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262for the preparation of PNAs. Further teaching of PNA compounds can befound in Nielsen et al., Science, 254:1497-1500, 1991. Each of theforegoing is incorporated by reference in its entirety.

2. Locked Nucleic Acids (LNAs)

Antisense oligomer compounds may also contain “locked nucleic acid”subunits (LNAs). “LNAs” are a member of a class of modifications calledbridged nucleic acid (BNA). BNA is characterized by a covalent linkagethat locks the conformation of the ribose ring in a C30-endo (northern)sugar pucker. For LNA, the bridge is composed of a methylene between the2′-0 and the 4′-C positions. LNA enhances backbone preorganization andbase stacking to increase hybridization and thermal stability.

The structures of LNAs can be found, for example, in Wengel, et al.,Chemical Communications (1998) 455; Tetrahedron (1998) 54:3607, andAccounts of Chem. Research (1999) 32:301); Obika, et al., TetrahedronLetters (1997) 38:8735; (1998) 39:5401, and Bioorganic MedicinalChemistry (2008) 16:9230, which are hereby incorporated by reference intheir entirety. A non-limiting example of an LNA is depicted below:

Compounds of the disclosure may incorporate one or more LNAs; in somecases, the compounds may be entirely composed of LNAs. Methods for thesynthesis of individual LNA nucleoside subunits and their incorporationinto oligomers are described, for example, in U.S. Pat. Nos. 7,572,582,7,569,575, 7,084,125, 7,060,809, 7,053,207, 7,034,133, 6,794,499, and6,670,461, each of which is incorporated by reference in its entirety.Typical intersubunit linkers include phosphodiester and phosphorothioatemoieties; alternatively, non-phosphorous containing linkers may beemployed. Further embodiments include an LNA containing compound whereeach LNA subunit is separated by a DNA subunit. Certain compounds arecomposed of alternating LNA and DNA subunits where the intersubunitlinker is phosphorothioate.

2′O,4′C-ethylene-bridged nucleic acids (ENAs) are another member of theclass of BNAs. A non-limiting example is depicted below:

ENA oligomers and their preparation are described in Obika et al.,Tetrahedron Ltt 38 (50): 8735, which is hereby incorporated by referencein its entirety. Compounds of the disclosure may incorporate one or moreENA subunits.

3. Phosphorothioates

“Phosphorothioates” (or S-oligos) are a variant of normal DNA in whichone of the nonbridging oxygens is replaced by a sulfur. A non-limitingexample of a phosphorothioate is depicted below:

The sulfurization of the internucleotide bond reduces the action ofendo- and exonucleases including 5′ to 3′ and 3′ to 5′ DNA POL 1exonuclease, nucleases S1 and P1, RNases, serum nucleases and snakevenom phosphodiesterase. Phosphorothioates are made by two principalroutes: by the action of a solution of elemental sulfur in carbondisulfide on a hydrogen phosphonate, or by the method of sulfurizingphosphite triesters with either tetraethylthiuram disulfide (TETD) or3H-1, 2-bensodithiol-3-one 1, 1-dioxide (BDTD) (see, e.g., Iyer et al.,J. Org. Chem. 55, 4693-4699, 1990, which are hereby incorporated byreference in their entirety). The latter methods avoid the problem ofelemental sulfur's insolubility in most organic solvents and thetoxicity of carbon disulfide. The TETD and BDTD methods also yieldhigher purity phosphorothioates.

4. Triclyclo-DNAs and Tricyclo-Phosphorothioate Nucleotides

Tricyclo-DNAs (tc-DNA) are a class of constrained DNA analogs in whicheach nucleotide is modified by the introduction of a cyclopropane ringto restrict conformational flexibility of the backbone and to optimizethe backbone geometry of the torsion angle γ. Homobasic adenine- andthymine-containing tc-DNAs form extraordinarily stable A-T base pairswith complementary RNAs. Tricyclo-DNAs and their synthesis are describedin International Patent Application Publication No. WO 2010/115993,which are hereby incorporated by reference in their entirety. Compoundsof the disclosure may incorporate one or more tricycle-DNA nucleotides;in some cases, the compounds may be entirely composed of tricycle-DNAnucleotides.

Tricyclo-phosphorothioate nucleotides are tricyclo-DNA nucleotides withphosphorothioate intersubunit linkages. Tricyclo-phosphorothioatenucleotides and their synthesis are described in International PatentApplication Publication No. WO 2013/053928, which are herebyincorporated by reference in their entirety. Compounds of the disclosuremay incorporate one or more tricycle-DNA nucleotides; in some cases, thecompounds may be entirely composed of tricycle-DNA nucleotides. Anon-limiting example of a tricycle-DNA/tricycle-phophothioate nucleotideis depicted below:

5. 2′ O-Methyl, 2′ O-MOE, and 2′-F Oligomers

“2′O-Me oligomer” molecules carry a methyl group at the 2′-OH residue ofthe ribose molecule. 2′-O-Me-RNAs show the same (or similar) behavior asDNA, but are protected against nuclease degradation. 2′-O-Me-RNAs canalso be combined with phosphothioate oligomers (PTOs) for furtherstabilization. 2′O-Me oligomers (phosphodiester or phosphothioate) canbe synthesized according to routine techniques in the art (see, e.g.,Yoo et al., Nucleic Acids Res. 32:2008-16, 2004, which is herebyincorporated by reference in its entirety). A non-limiting example of a2′ O-Me oligomer is depicted below:

2′ O-Me oligomers may also comprise a phosphorothioate linkage (2′ O-Mephosphorothioate oligomers). 2′ O-Methoxyethyl Oligomers (2′-O MOE),like 2′ O-Me oligomers, carry a methoxyethyl group at the 2′-OH residueof the ribose molecule and are discussed in Martin et al., Helv. Chim.Acta, 78, 486-504, 1995, which are hereby incorporated by reference intheir entirety. A non-limiting example of a 2′ O-MOE nucleotide isdepicted below:

In contrast to the preceding alkylated 2′OH ribose derivatives, 2-fluorooligomers have a fluoro radical in at the 2′ position in place of the2′OH. A non-limiting example of a 2′-F oligomer is depicted below:

2′-fluoro oligomers are further described in WO 2004/043977, which ishereby incorporated by reference in its entirety. Compounds of thedisclosure may incorporate one or more 2′O-Methyl, 2′ O-MOE, and 2′-Fsubunits and may utilize any of the intersubunit linkages describedhere. In some instances, a compound of the disclosure could be composedof entirely 2′O-Methyl, 2′ O-MOE, or 2′-F subunits. One embodiment of acompound of the disclosure is composed entirely of 2′O-methyl subunits.

6. 2′-O-[2-(N-methylcarbamoyl)ethyl] Oligonucleotides (MCEs)

MCEs are another example of 2′O modified ribonucleosides useful in thecompounds of the disclosure. Here, the 2′OH is derivatized to a2-(N-methylcarbamoyl)ethyl moiety to increase nuclease resistance. Anon-limiting example of an MCE oligomer is depicted below:

MCEs and their synthesis are described in Yamada et al., J. Org. Chem.,76(9):3042-53, which is hereby incorporated by reference in itsentirety. Compounds of the disclosure may incorporate one or more MCEsubunits.

7. Stereo Specific Oligomers

Stereo specific oligomers are those which the stereo chemistry of eachphosphorous-containing linkage is fixed by the method of synthesis suchthat a substantially pure single oligomer is produced. A non-limitingexample of a stereo specific oligomer is depicted below:

In the above example, each phosphorous of the oligomer has the samestereo chemistry. Additional examples include the oligomers describedabove. For example, LNAs, ENAs, Tricyclo-DNAs, MCEs, 2′ O-Methyl, 2′O-MOE, 2′-F, and morpholino-based oligomers can be prepared withstereo-specific phosphorous-containing internucleoside linkages such as,for example, phosphorothioate, phosphodiester, phosphoramidate,phosphorodiamidate, or other phosphorous-containing internucleosidelinkages. Stereo specific oligomers, methods of preparation, chirolcontrolled synthesis, chiral design, and chiral auxiliaries for use inpreparation of such oligomers are detailed, for example, inWO2015107425, WO2015108048, WO2015108046, WO2015108047, WO2012039448,WO2010064146, WO2011034072, WO2014010250, WO2014012081, WO20130127858,and WO2011005761, each of which is hereby incorporated by reference inits entirety.

8. Morpholino-based Oligomers

Morpholino-based oligomers refer to an oligomer comprising morpholinosubunits supporting a nucleobase and, instead of a ribose, contains amorpholine ring. Exemplary internucleoside linkages include, forexample, phosphoramidate or phosphorodiamidate internucleoside linkagesjoining the morpholine ring nitrogen of one morpholino subunit to the 4′exocyclic carbon of an adjacent morpholino subunit. Each morpholinosubunit comprises a purine or pyrimidine nucleobase effective to bind,by base-specific hydrogen bonding, to a base in an oligonucleotide.

Morpholino-based oligomers (including antisense oligomers) are detailed,for example, in U.S. Pat. Nos. 5,698,685; 5,217,866; 5,142,047;5,034,506; 5,166,315; 5,185,444; 5,521,063; 5,506,337 and pending U.S.patent application Ser. Nos. 12/271,036; 12/271,040; and PCT PublicationNo. WO/2009/064471 and WO/2012/043730 and Summerton et al. 1997,Antisense and Nucleic Acid Drug Development, 7, 187-195, which arehereby incorporated by reference in their entirety. Within the oligomerstructure, the phosphate groups are commonly referred to as forming the“internucleoside linkages” of the oligomer. The naturally occurringinternucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiesterlinkage. A “phosphoramidate” group comprises phosphorus having threeattached oxygen atoms and one attached nitrogen atom, while a“phosphorodiamidate” group comprises phosphorus having two attachedoxygen atoms and two attached nitrogen atoms. In the uncharged or thecationic intersubunit linkages of morpholino-based oligomers describedherein, one nitrogen is always pendant to the backbone chain. The secondnitrogen, in a phosphorodiamidate linkage, is typically the ringnitrogen in a morpholine ring structure.

“PMO-X” refers to phosphorodiamidate morpholino-based oligomers having aphosphorus atom with (i) a covalent bond to the nitrogen atom of amorpholine ring and (ii) a second covalent bond to the ring nitrogen ofa 4-aminopiperdin-1-yl (i.e., APN) or a derivative of4-aminopiperdin-1-yl. Exemplary and non-limiting PMO-X oligomers aredisclosed in PCT Application No. PCT/US2011/38459 and PCT PublicationNo. WO 2013/074834, which are hereby incorporated by reference in theirentirety. PMO-X includes “PMO-apn” or “APN,” which refers to a PMO-Xoligomer which comprises at least one internucleoside linkage where aphosphorus atom is linked to a morpholino group and to the ring nitrogenof a 4-aminopiperdin-1-yl (i.e., APN). In specific embodiments, anantisense oligomer comprising a targeting sequence comprises at leastone APN-containing linkage or APN derivative-containing linkage. Variousembodiments include morpholino-based oligomers that have about 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 100% APN/APN derivative-containing linkages, where theremaining linkages (if less than 100%) are uncharged linkages, e.g.,about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 of the total internucleoside linkagesare APN/APN derivative-containing linkages.

In some embodiments, the antisense oligomer is a compound of formula(I):

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   each Nu is a nucleobase which taken together form a targeting        sequence;    -   Z is an integer from 8 to 38;    -   each Y is independently selected from O and —NR⁴, wherein each        R⁴ is independently selected from H, C₁-C₆ alkyl, aralkyl,        —C(═NH)NH₂, —C(O)(CH₂)_(n)NR⁵C(═NH)NH₂,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NR⁵C(═NH)NH₂, and G, wherein R⁵ is        selected from H and C₁-C₆ alkyl and n is an integer from 1 to 5;    -   T is selected from OH and a moiety of the formula:

-   -   -   wherein:        -   A is selected from —OH, —N(R⁷)₂, and R¹ wherein each R⁷ is            independently selected from H and C₁-C₆ alkyl, and        -   R⁶ is selected from OH, —N(R⁹)CH₂C(O)NH₂, and a moiety of            the formula:

-   -   -   wherein:            -   R⁹ is selected from H and C₁-C₆ alkyl; and            -   R¹⁰ is selected from G, —C(O)—R¹¹OH, acyl, trityl,                4-methoxytrityl,            -   —C(═NH)NH₂, —C(O)(CH₂)_(m)NR¹²C(═NH)NH₂, and            -   —C(O)(CH₂)₂NHC(O)(CH₂)₅NR¹²C(═NH)NH₂, wherein:            -   m is an integer from 1 to 5,            -   R¹¹ is of the formula —(O-alkyl)_(y)- wherein y is an                integer from 3 to 10 and                -   each of the y alkyl groups is independently selected                    from C₂-C₆ alkyl; and            -   R¹² is selected from H and C₁-C₆ alkyl;

    -   each instance of R¹ is independently selected from:        -   —N(R¹³)₂, wherein each R¹³ is independently selected from H            and C₁-C₆ alkyl;        -   a moiety of formula (II):

-   -   -   -   wherein:                -   R¹⁵ is selected from H, G, CL-C6 alkyl, —C(═NH)NH₂,                -   —C(O)(CH₂)_(q)NR¹⁸C(═NH)NH₂, and                -   —C(O)(CH₂)₂NHC(O)(CH₂)₅NR¹⁸C(NH)NH₂, wherein:                -    R¹⁸ is selected from H and C₁-C₆ alkyl; and                -    q is an integer from 1 to 5, and                -   each R¹⁷ is independently selected from H and                    methyl; and

        -   a moiety of formula (III):

-   -   -   -   wherein:                -   R¹⁹ is selected from H, C₁-C₆ alkyl, —C(═NH)NH₂,                    —C(O)(CH₂)_(r)NR²²C(═NH)NH₂,                -   —C(O)CH(NH₂)(CH₂)₃NHC(═NH)NH₂,                -   —C(O)(CH₂)₂NHC(O)(CH₂)₅NR²²C(═NH)NH₂,                -   —C(O)CH(NH₂)(CH₂)₄NH₂ and G, wherein:                -    R²² is selected from H and C₁-C₆ alkyl; and                -    r is an integer from 1 to 5, and                -   R²⁰ is selected from H and C₁-C₆ alkyl; or                -   R¹⁹ and R²⁰ together with the nitrogen atom to which                    they are attached form a heterocyclic or heteroaryl                    ring having from 5 to 7 ring atoms and optionally                    containing an additional heteroatom selected from                    oxygen, nitrogen, and sulfur; and

    -   R² is selected from H, G, acyl, trityl, 4-methoxytrityl,        benzoyl, stearoyl, C₁-C₆ alkyl, —C(═NH)NH₂, —C(O)—R²³,        —C(O)(CH₂)_(s)NR²⁴C(═NH)NH₂,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NR²⁴C(═NH)NH₂,        —C(O)CH(NH₂)(CH₂)₃NHC(═NH)NH₂, and a moiety of the formula:

-   -   -   wherein,            -   R²³ is of the formula —(O-alkyl)_(v)-OH wherein v is an                integer from 3 to 10 and each of the v alkyl groups is                independently selected from C₂-C₆ alkyl; and            -   R²⁴ is selected from H and C₁-C₆ alkyl;            -   s is an integer from 1 to 5;            -   L is selected from —C(O)(CH₂)₆C(O)— and                —C(O)(CH₂)₂S2(CH₂)₂C(O)—; and            -   each R²⁵ is of the formula —(CH₂)₂OC(O)N(R²⁶)₂ wherein                each R²⁶ is of the formula —(CH₂)₆NHC(═NH)NH₂,

    -   wherein G is a cell penetrating peptide (“CPP”) and linker        moiety selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus, and    -   wherein G may be present in one occurrence or is absent.

In some embodiments, R² is a moiety of the formula:

where L is selected from —C(O)(CH₂)₆C(O)— or —C(O)(CH₂)₂S₂(CH₂)₂C(O)—,and

-   -   and each R²⁵ is of the formula —(CH₂)₂OC(O)N(R²⁶)₂ wherein each        R²⁶ is of the formula —(CH₂)₆NHC(═NH)NH₂. Such moieties are        further described in U.S. Pat. No. 7,935,816 incorporated herein        by reference in its entirety.

In certain embodiments, R² may comprise either moiety depicted below:

In certain embodiments, each R¹ is —N(CH₃)₂. In some embodiments, about50-90% of the R₁ groups are dimethylamino (i.e. —N(CH₃)₂). In certainembodiments, about 66% of the R₁ groups are dimethylamino.

In some non-limiting embodiments, each R¹ is —N(CH₃)₂ and X is selectedfrom uracil (U) or thymine (T).

In some embodiments of the disclosure, R₁ may be selected from:

In some embodiments, at least one R¹ is:

In certain embodiments, T is selected from:

-   -   Y is O at each occurrence. In some embodiments. R² is selected        from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, T is selected from:

-   -   Y is O at each occurrence at each occurrence and R² is G.

In some embodiments, T is of the formula:

-   -   R⁶ is of the formula:

-   -   Y is O at each occurrence and R² is G.

In certain embodiments, T is of the formula:

-   -   Y is O at each occurrence and R² is G. In some embodiments, T is        of the formula:

-   -   Y is O at each occurrence, each R¹ is —N(CH₃)₂, and R² is G.

In certain embodiments, T is of the formula:

-   -   and Y is O at each occurrence. In some embodiments, T is of the        formula:

-   -   Y is O at each occurrence, each R¹ is —N(CH₃)₂, and R² is        acetyl.

In certain embodiments, T is of the formula:

Y is O at each occurrence, each R¹ is —N(CH₃)₂, and R² is H.

In some embodiments, R² is selected from H, acyl, trityl,4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, R² is selected from H or G. In a particularembodiment, R² is G. In some embodiments, R² is H or acyl. In someembodiments, each R¹ is —N(CH₃)₂. In some embodiments, at least oneinstance of R¹ is —N(CH₃)₂. In certain embodiments, each instance of R¹is —N(CH₃)₂.

In some embodiments, G is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl.

In another aspect, the antisense oligomer is a compound of formula (Ia):

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   each Nu is a nucleobase which taken together form a targeting        sequence;    -   Z is an integer from about 13 to about 38;    -   each Y is independently selected from O and —NR⁴, wherein each        R⁴ is independently selected from H, C₁-C₆ alkyl, aralkyl,        —C(═NH)NH₂, —C(O)(CH₂)_(n)NR⁵C(═NH)NH₂,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NR⁵C(═NH)NH₂, and G, wherein R⁵ is        selected from H and C₁-C₆ alkyl and n is an integer from 1 to 5;    -   T is selected from OH and a moiety of the formula:

-   -   -   wherein:        -   A is selected from —OH, —N(R⁷)₂, and R¹ wherein:            -   each R⁷ is independently selected from H and C₁-C₆                alkyl, and            -   R⁶ is selected from OH, —N(R⁹)CH₂C(O)NH₂, and a moiety                of the formula:

-   -   -   wherein:            -   R⁹ is selected from H and C₁-C₆ alkyl, and            -   R¹⁰ is selected from G, —C(O)—R¹¹OH, acyl, trityl,                4-methoxytrityl,            -   —C(═NH)NH₂, —C(O)(CH₂)_(m)NR¹²C(═NH)NH₂, and            -   —C(O)(CH₂)₂NHC(O)(CH₂)₅NR¹²C(═NH)NH₂, wherein:                -   m is an integer from 1 to 5,                -   R¹¹′ is of the formula —(O-alkyl)_(y)- wherein y is                    an integer from 3 to 10 and                -    each of the y alkyl groups is independently                    selected from C₂-C₆ alkyl; and                -   R¹² is selected from H and C₁-C₆ alkyl;

    -   each instance of R¹ is independently selected from:        -   —N(R¹³)₂, wherein each R¹³ is independently selected from H            and C₁-C₆ alkyl; a moiety of formula (II):

-   -   -   -   wherein:                -   R¹⁵ is selected from H, G, C₁-C₆ alkyl, —C(═NH)NH₂,                -   —C(O)(CH₂)_(q)NR¹⁸C(═NH)NH₂, and                -   —C(O)(CH₂)₂NHC(O)(CH₂)₅NR¹⁸C(═NH)NH₂, wherein:                -    R¹⁸ is selected from H and C₁-C₆ alkyl; and                -    q is an integer from 1 to 5; and                -   each R¹⁷ is independently selected from H and                    methyl; and

        -   a moiety of formula (III):

-   -   -   -   wherein:                -   R¹⁹ is selected from H, C₁-C₆ alkyl, —C(═NH)NH₂,                    —C(O)(CH₂)_(r)NR²²C(═NH)NH₂,                -    —C(O)CH(NH₂)(CH₂)₃NHC(═NH)NH₂,                -    —C(O)(CH₂)₂NHC(O)(CH₂)₅NR²²C(═NH)NH₂,                -    —C(O)CH(NH₂)(CH₂)₄NH₂ and G, wherein:                -    R²² is selected from H and C₁-C₆ alkyl; and                -    r is an integer from 1 to 5, and                -   R²⁰ is selected from H and C₁-C₆ alkyl; or                -   R¹⁹ and R²⁰ together with the nitrogen atom to which                    they are attached form a heterocyclic or heteroaryl                    ring having from 5 to 7 ring atoms and optionally                    containing an additional heteroatom selected from                    oxygen, nitrogen, and sulfur; and

    -   R² is selected from H, G, acyl, trityl, 4-methoxytrityl,        benzoyl, stearoyl, C₁-C₆ alkyl, —C(═NH)NH₂, —C(O)—R²³,        —C(O)(CH₂)_(s)NR²⁴C(═NH)NH₂,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NR²⁴C(═NH)NH₂,        —C(O)CH(NH₂)(CH₂)₃NHC(═NH)NH₂, and a moiety of the formula:

-   -   -   wherein,            -   R²³ is of the formula —(O-alkyl)_(v)-OH wherein v is an                integer from 3 to 10 and each of the v alkyl groups is                independently selected from C₂-C₆ alkyl; and            -   R²⁴ is selected from H and C₁-C₆ alkyl;            -   s is an integer from 1 to 5;            -   L is selected from —C(O)(CH₂)₆C(O)— and                —C(O)(CH₂)₂S2(CH₂)₂C(O)—; and            -   each R²⁵ is of the formula —(CH₂)₂OC(O)N(R²⁶)₂ wherein                each R²⁶ is of the formula —(CH₂)₆NHC(═NH)NH₂,

    -   wherein G is a cell penetrating peptide (“CPP”) and linker        moiety comprising the formula —C(O)CH₂NH—CPP, where CPP is of        the formula:

-   -   wherein R^(a) is H or acyl, and    -   wherein G may be present in one occurrence or is absent.

In certain embodiments, T is selected from:

and

-   -   Y is O at each occurrence. In some embodiments, R² is selected        from H, G, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, T is selected from:

Y is O at each occurrence and R² is G.

In some embodiments, T is of the formula:

-   -   R⁶ is of the formula:

-   -   Y is O at each occurrence and R² is G.

In certain embodiments. T is of the formula:

-   -   Y is O at each occurrence and R² is G. In some embodiments, T is        of the formula:

-   -   Y is O at each occurrence, each R¹ is —N(CH₃)₂, and R² is G.

In certain embodiments, T is of the formula:

-   -   and Y is O at each occurrence. In some embodiments, T is of the        formula:

-   -   Y is O at each occurrence, each R¹ is —N(CH₃)₂, and R² is        acetyl.

In certain embodiments, T is of the formula:

Y is O at each occurrence, each R¹ is —N(CH₃)₂, and R² is H.

In some embodiments, R² is selected from H, acyl, trityl,4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, R² is selected from H or G. In a particularembodiment, R² is G. In some embodiments, R² is H or acyl. In someembodiments, each R′ is —N(CH₃)₂. In some embodiments, at least oneinstance of R¹ is —N(CH₃)₂. In certain embodiments, each instance of R′is —N(CH₃)₂.

In some embodiments, R^(a) is acetyl.

In embodiments including, for example, embodiments of the antisenseoligomers of formula (I) and (Ia), the targeting sequence iscomplementary to a target region within intron 1 of a pre-mRNA of thehuman alpha glucosidase (GAA) gene. In embodiments including, forexample, embodiments of the antisense oligomers of formula (I) and (Ia),the targeting sequence is complementary to a target region within intron1 of a pre-mRNA of the human alpha glucosidase (GAA) gene, wherein thetarget region comprises at least one additional nucleobase compared tothe targeting sequence, wherein the at least one additional nucleobasehas no complementary nucleobase in the targeting sequence, and whereinthe at least one additional nucleobase is internal to the target region.

In embodiments, a sequence with 100% complementarity is selected and oneor more nucleobases is removed (or alternately are synthesized with oneor more missing nucleobases) so that the resulting sequence has one ormore missing nucleobases than its natural complement in the targetregion. With the exception of the portion where one or more nucleobasesare removed, it is contemplated that the remaining portions are 100%complementary. However, it is within the scope of this invention thatdecreased levels of complementarity could be present.

In certain embodiments, the antisense oligomer of the disclosure is acompound of formula (IVa):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each Nu is a nucleobase which taken together forms a targeting        sequence;    -   Z is an integer from 8 to 38;    -   T is selected from OH and a moiety of the formula:

-   -   -   wherein:        -   A is selected from —OH, —N(R⁷)₂R⁸, and R¹ wherein:            -   each R⁷ is independently selected from H and C₁-C₆                alkyl, and            -   R⁸ is selected from an electron pair and H, and        -   R⁶ is selected from OH, —N(R⁹)CH₂C(O)NH₂, and a moiety of            the formula:

-   -   -   wherein:            -   R⁹ is selected from H and C₁-C₆ alkyl; and            -   R¹⁰ is selected from —C(O)—R¹¹OH, acyl, trityl,                4-methoxytrityl,            -   —C(═NH)NH₂, —C(O)(CH₂)_(m)NR¹²C(═NH)NH₂, and            -   —C(O)(CH₂)₂NHC(O)(CH₂)₅NR¹²C(═NH)NH₂, wherein:                -   m is an integer from 1 to 5,                -   R¹¹ is of the formula —(O-alkyl)_(y)- wherein y is                    an integer from 3 to 10 and                -    each of the y alkyl groups is independently                    selected                -    from C₂-C₆ alkyl; and            -   R¹² is selected from H and C₁-C₆ alkyl;

    -   each instance of R¹ is independently —N(R¹³)₂R¹⁴, wherein each        R¹³ is independently selected from H and C₁-C₆ alkyl, and R¹⁴ is        selected from an electron pair and H; and

    -   R² is selected from H, acyl, trityl, 4-methoxytrityl, benzoyl,        stearoyl, and C₁-C₆ alkyl.

In certain embodiments. T is selected from:

and

-   -   Y is O at each occurrence. In some embodiments, R² is selected        from H, acyl, trityl, 4-methoxytrityl, benzoyl, and stearoyl.

In various embodiments, T is selected from:

In some embodiments, T is of the formula:

and

-   -   R⁶ is of the formula:

In certain embodiments, T is of the formula:

In some embodiments, R² is H, trityl, or acyl. In some embodiments, atleast one instance of R¹ is —N(CH₃)₂. In some embodiments, each R¹ is—N(CH₃)₂.

In certain embodiments, the antisense oligomer of the disclosure is acompound of formula (IVb):

or a pharmaceutically acceptable salt thereof, where:

-   -   each Nu is a nucleobase which taken together forms a targeting        sequence;    -   Z is an integer from 8 to 38;    -   T is selected from a moiety of the formula:

wherein R³ is selected from H and C₁-C₆ alkyl;

-   -   each instance of R¹ is independently —N(R⁴)₂, wherein each R⁴ is        independently selected from H and C₁-C₆ alkyl; and    -   R² is selected from H, acyl, trityl, 4-methoxytrityl, benzoyl,        stearoyl, and C₁-C₆ alkyl.

In various embodiments, R² is selected from H or acyl. In someembodiments, R² is H.

In certain embodiments, T is of the formula:

and

-   -   R² is hydrogen.

In certain embodiments, the antisense oligomer of the disclosure is acompound of formula (IVc):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each Nu is a nucleobase which taken together form a targeting        sequence;    -   Z is an integer from 8 to 38,    -   each Y is O;    -   each R¹ is independently selected from the group consisting of

wherein at least one R¹ is —N(CH₃)₂.

In some embodiments, X is selected from uracil (U) or thymine (T). Insome embodiments, each R¹ is —N(CH₃)₂.

In certain embodiments, the antisense oligomer is a compound of formula(V):

or a pharmaceutically acceptable salt thereof, wherein:

-   -   each Nu is a nucleobase which taken together form a targeting        sequence; and    -   Z is an integer from 8 to 38.

In some embodiments including, for example, embodiments of the antisenseoligomers of formula (IVa), (IVb), (IVc) and (V), the targeting sequenceis complementary to a target region that comprises an exon targetassociated with Duchenne muscular dystrophy. In certain embodiments, thetargeting sequence is complementary to a target sequence that comprisesexon 44 in the processing of human dystrophin pre-processed mRNA.Further, in certain embodiments, a sequence with 100% complementarity isselected and one or more nucleobases is removed (or alternately aresynthesized with one or more missing nucleobases) so that the resultingsequence has one or more missing nucleobases than its natural complementin the target region. With the exception of the portion where one ormore nucleobases are removed, it is contemplated that the remainingportions are 100% complementary. However, it is within the scope of thisinvention that decreased levels of complementarity could be present. Inembodiments, at least one nucleobase is removed when the targetingsequence would otherwise include a string of three or four of moreidentical contiguous nucleobases or a biological palindrome sequence.

In some embodiments including, for example, embodiments of the antisenseoligomers of formula (IVa), (IVb), (IVc) and (V), the targeting sequenceis complementary to a target region that comprises an exon targetassociated with spinal muscular atrophy. In certain embodiments, thetargeting sequence is complementary to a target sequence that comprisesa region adjacent to exon 7 in the processing of human SMN2pre-processed mRNA. Further, in certain embodiments, a sequence with100% complementarity is selected and one or more nucleobases is removed(or alternately are synthesized with one or more missing nucleobases) sothat the resulting sequence has one or more missing nucleobases than itsnatural complement in the target region. With the exception of theportion where one or more nucleobases are removed, it is contemplatedthat the remaining portions are 100% complementary. However, it iswithin the scope of this invention that decreased levels ofcomplementarity could be present. In embodiments, at least onenucleobase is removed when the targeting sequence would otherwiseinclude a string of three or four of more identical contiguousnucleobases or a biological palindrome sequence.

In some embodiments including, for example, embodiments of the antisenseoligomers of formula (IVa), (IVb), (IVc) and (V), the targeting sequenceis complementary to a target region within intron 1 of a pre-mRNA of thehuman alpha glucosidase (GAA) gene. In various embodiments including,for example, embodiments of the antisense oligomers of formula (IVa),(IVb), (IVc) and (V), the targeting sequence is complementary to atarget region associated with exon 2 of a pre-mRNA of the human alphaglucosidase (GAA) gene, wherein the target region comprises at least oneadditional nucleobase compared to the targeting sequence, wherein the atleast one additional nucleobase has no complementary nucleobase in thetargeting sequence, and wherein the at least one additional nucleobaseis internal to the target region. Further, in certain embodiments, asequence with 100% complementarity is selected and one or morenucleobases is removed (or alternately are synthesized with one or moremissing nucleobases) so that the resulting sequence has one or moremissing nucleobases than its natural complement in the target region.With the exception of the portion where one or more nucleobases areremoved, it is contemplated that the remaining portions are 100%complementary. However, it is within the scope of this invention thatdecreased levels of complementarity could be present. In embodiments, atleast one nucleobase is removed when the targeting sequence wouldotherwise include a string of three or four of more identical contiguousnucleobases or a biological palindrome sequence.

In various aspects, the antisense oligomers comprise the deletionsequence of any one of SEQ ID NOs: 1-128, wherein at least onenucleobase in any one of SEQ ID NOs: 1-128 has been deleted. In variousembodiments, the oligonucleotide comprisesCTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 69; Eteplirsen);GTTGCCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 70; Golodirsen); orCAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 71; Casimersen). In variousembodiments, the deletion sequence comprises any one of SEQ ID NOs:69-71, wherein at least one nucleobase in any one of SEQ ID NOs: 72-74has been deleted. In embodiments, the at least one nucleobase that hasbeen deleted is internal to the sequence of any one of SEQ ID NOs:69-71.

In certain embodiments, the antisense oligomer is a compound of formula(VI):

-   -   or a pharmaceutically acceptable salt thereof,    -   where each Nu is a nucleobase which taken together forms a        targeting sequence;    -   Z is an integer from 8 to 38;    -   T is selected from:

-   -   each R¹ is independently selected from the group consisting of

-   -   R² is selected from H, G, acyl, trityl, 4-methoxytrityl,        benzoyl, and stearoyl,    -   wherein G is a cell penetrating peptide (“CPP”) and linker        moiety selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus, and    -   wherein T is

or R² is G.

In certain embodiments. T is of the formula:

-   -   and R² is G. In certain embodiments, at least one occurrence of        R¹ is —N(CH₃)₂. In some embodiments, each occurrence of R₁ is        —N(CH₃)₂. In some embodiments, T is of the formula:

In certain embodiments, at least one occurrence of R¹ is N(CH₃)₂. Insome embodiments, each occurrence of R¹ is N(CH₃)₂.

In some embodiments, T is of the formula:

-   -   R² is G, and each occurrence of R¹ is —N(CH₃)₂.

In certain embodiments, R² is selected from H, acetyl, trityl,4-methoxytrityl, benzoyl, and stearoyl and T is of the formula:

In various embodiments, R² is acetyl. In certain embodiments, at leastone occurrence of R¹ is —N(CH₃)₂. In some embodiments, each occurrenceof R¹ is —N(CH₃)₂.

In various embodiments, R² is selected from H, acyl, trityl,4-methoxytrityl, benzoyl, and stearoyl.

In certain embodiments, R2 is acetyl, T is of the formula:

and each occurrence of R¹ is —N(CH₃)₂.

In some embodiments, wherein G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In some embodiments, the CPP is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In certain embodiments, the antisense oligomer is a compound of formula(VII):

-   -   or a pharmaceutically acceptable salt thereof,    -   where each Nu is a nucleobase which taken together forms a        targeting sequence;    -   Z is an integer from 8 to 38;    -   T is selected from:

-   -   each R¹ is —N(R⁴)₂ wherein each R⁴ is independently C₁-C₆ alkyl;        and    -   R² is selected from H, G, acyl, trityl, 4-methoxytrityl,        benzoyl, and stearoyl,    -   wherein G is a cell penetrating peptide (“CPP”) and linker        moiety selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus, and    -   wherein T is

or R² is G.

In some embodiments, at least one instance of R¹ is —N(CH₃)₂. In certainembodiments, each instance of R¹ is —N(CH₃)₂.

In certain embodiments, T is of the formula:

and R² is G. In some embodiments, at least one instance of R¹ is—N(CH₃)₂. In certain embodiments, each instance of R¹ is —N(CH₃)₂.

In various embodiments, G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

wherein R^(a) is selected from H, acetyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In certain embodiments, the antisense oligomer is a compound of formula(VIIa):

-   -   or a pharmaceutically acceptable salt thereof,    -   where each Nu is a nucleobase which taken together forms a        targeting sequence;    -   Z is an integer from 8 to 38;    -   T is selected from:

-   -   each instance of R¹ is —N(R⁴)₂ wherein each R⁴ is independently        C₁-C₆ alkyl; and    -   G is a cell penetrating peptide (“CPP”) and linker moiety        selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus.

In some embodiments, at least one instance of R¹ is —N(CH₃)₂. In certainembodiments, each instance of R¹ is —N(CH₃)₂.

In some embodiments, G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In various embodiments, each instance of R is —N(CH₃)₂, G is of theformula:

and

-   -   R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl. In various embodiments,        each instance of R¹ is —N(CH₃)₂, the CPP is of the formula:

and

-   -   R^(a) is acetyl.

In various aspects, an antisense oligonucleotide of the disclosureincludes a compound of formula (VIIb):

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   where each Nu is a nucleobase which taken together forms a        targeting sequence;    -   Z is an integer from 8 to 38;    -   each instance of R¹ is —N(R⁴)₂ wherein each R⁴ is independently        C₁-C₆ alkyl; and    -   G is a cell penetrating peptide (“CPP”) and linker moiety        selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus.

In some embodiments, at least one instance of R¹ is —N(CH₃)₂. In certainembodiments, each instance of R¹ is —N(CH₃)₂.

In some embodiments, G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In various embodiments, each instance of R¹ is —N(CH₃)₂, G is of theformula:

and

-   -   R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl. In various embodiments,        each instance of R¹ is —N(CH₃)₂, the CPP is of the formula:

and

-   -   R^(a) is acetyl.

In various aspects, an antisense oligonucleotide of the disclosureincludes a compound of formula (VIIc):

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   where each Nu is a nucleobase which taken together forms a        targeting sequence;    -   Z is an integer from 8 to 38; and    -   G is a cell penetrating peptide (“CPP”) and linker moiety        selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus.

In some embodiments, G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In various embodiments, G is of the formula:

and

-   -   R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl. In various embodiments,        the CPP is of the formula:

and

-   -   R^(a) is acetyl.

In various aspects, an antisense oligomer of the disclosure is acompound of formula (VIId):

-   -   wherein:

-   -   each Nu is a nucleobase which taken together forms a targeting        sequence;    -   Z is an integer from 8 to 38;    -   each instance of R¹ is —N(R⁴)₂ wherein each R⁴ is independently        C₁-C₆ alkyl; and    -   R² is selected from H, trityl, 4-methoxytrityl, acetyl, benzoyl,        and stearoyl; and    -   G is a cell penetrating peptide (“CPP”) and linker moiety        selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus.

In some embodiments, at least one instance of R1 is —N(CH₃)₂. In certainembodiments, each instance of R¹ is —N(CH₃)₂.

In some embodiments, G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In various embodiments, each instance of R is —N(CH₃)₂, G is of theformula:

and

-   -   R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl. In various embodiments,        each instance of R¹ is —N(CH₃)₂, the CPP is of the formula:

and

-   -   R^(a) is acetyl.

In various aspects, an antisense oligonucleotide of the disclosureincludes a compound of formula (VIIe):

-   -   or a pharmaceutically acceptable salt thereof, wherein:    -   each Nu is a nucleobase which taken together forms a targeting        sequence;    -   Z is an integer from 8 to 38;    -   R² is selected from H, trityl, 4-methoxytrityl, acetyl, benzoyl,        and stearoyl; and    -   G is a cell penetrating peptide (“CPP”) and linker moiety        selected from —C(O)(CH₂)₅NH—CPP, —C(O)(CH₂)₂NH—CPP,        —C(O)(CH₂)₂NHC(O)(CH₂)₅NH—CPP, —C(O)CH₂NH—CPP, and:

-   -   wherein the CPP is attached to the linker moiety by an amide        bond at the CPP carboxy terminus.

In some embodiments, G is of the formula:

wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl. In someembodiments, R^(a) is acetyl.

In various embodiments, G is of the formula:

and

-   -   R^(a) is acetyl.

In certain embodiments, the CPP is of the formula:

-   -   wherein R^(a) is selected from H, acyl, benzoyl, and stearoyl.        In some embodiments, R^(a) is acetyl. In various embodiments,        the CPP is of the formula:

and

-   -   R^(a) is acetyl.

In various embodiments including, for example, embodiments of theantisense oligomers of formula (VI), (VII), (VIIa), (VIIb), (VIIc),(VIId), (VIIe), and (VIII), the targeting sequence is complementary to atarget region that comprises an exon target associated with Duchennemuscular dystrophy. In certain embodiments, the targeting sequence iscomplementary to a target sequence that comprises exon 44 in theprocessing of human dystrophin pre-processed mRNA. Further, in certainembodiments, a sequence with 100% complementarity is selected and one ormore nucleobases is removed (or alternately are synthesized with one ormore missing nucleobases) so that the resulting sequence has one or moremissing nucleobases than its natural complement in the target region.With the exception of the portion where one or more nucleobases areremoved, it is contemplated that the remaining portions are 100%complementary. However, it is within the scope of this invention thatdecreased levels of complementarity could be present. In embodiments, atleast one nucleobase is removed when the targeting sequence wouldotherwise include a string of three or four of more identical contiguousnucleobases or a biological palindrome sequence.

In various embodiments including, for example, embodiments of theantisense oligomers of formula (VI), (VII), (VIIa), (VIIb), (VIIc),(VIId), (VIIe), and (VIII), the targeting sequence is complementary to atarget region that comprises an exon target associated with spinalmuscular atrophy. In certain embodiments, the targeting sequence iscomplementary to a target sequence that comprises a region adjacent toexon 7 in the processing of human SMN2 pre-processed mRNA. Further, incertain embodiments, a sequence with 100% complementarity is selectedand one or more nucleobases is removed (or alternately are synthesizedwith one or more missing nucleobases) so that the resulting sequence hasone or more missing nucleobases than its natural complement in thetarget region. With the exception of the portion where one or morenucleobases are removed, it is contemplated that the remaining portionsare 100% complementary. However, it is within the scope of thisinvention that decreased levels of complementarity could be present. Inembodiments, at least one nucleobase is removed when the targetingsequence would otherwise include a string of three or four of moreidentical contiguous nucleobases or a biological palindrome sequence.

In various embodiments including, for example, embodiments of theantisense oligomers of formula (VI), (VII), (VIIa), (VIIb), (VIIc),(VIId), (VIIe), and (VIII), the targeting sequence is complementary to atarget region within intron 1 of a pre-mRNA of the human alphaglucosidase (GAA) gene. In various embodiments including, for example,embodiments of the antisense oligomers of formula (VI), (VII), (VIIa),(VIIb), (VIIc), (VIId), (VIIe), and (VIII), the targeting sequence iscomplementary to a target region associated with exon 2 of a pre-mRNA ofthe human alpha glucosidase (GAA) gene, wherein the target regioncomprises at least one additional nucleobase compared to the targetingsequence, wherein the at least one additional nucleobase has nocomplementary nucleobase in the targeting sequence, and wherein the atleast one additional nucleobase is internal to the target region.Further, in certain embodiments, a sequence with 100% complementarity isselected and one or more nucleobases is removed (or alternately aresynthesized with one or more missing nucleobases) so that the resultingsequence has one or more missing nucleobases than its natural complementin the target region. With the exception of the portion where one ormore nucleobases are removed, it is contemplated that the remainingportions are 100% complementary. However, it is within the scope of thisinvention that decreased levels of complementarity could be present. Inembodiments, at least one nucleobase is removed when the targetingsequence would otherwise include a string of three or four of moreidentical contiguous nucleobases or a biological palindrome sequence.

In various aspects, the antisense oligomers comprise the deletionsequence of any one of SEQ ID NOs: 1-128, wherein at least onenucleobase in any one of SEQ ID NOs: 1-128 has been deleted. In variousembodiments, the oligonucleotide comprisesCTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 69; Eteplirsen);GTTGCCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 70; Golodirsen); orCAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 71; Casimersen).

In some embodiments of any of the antisense oligomers, methods, orcompositions described herein, Z is an integer from 8 to 28, from 15 to38, 15 to 28, 8 to 25, from 15 to 25, from 10 to 38, from 10 to 25, from12 to 38, from 12 to 25, from 14 to 38, or from 14 to 25. In someembodiments of any of the antisense oligomers, methods, or compositionsdescribed herein, Z is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38. In someembodiments of any of the antisense oligomers, methods, or compositionsdescribed herein, Z is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, or 28. In some embodiments of any of the antisenseoligomers, methods, or compositions described herein, Z is 15, 16, 17,18, 19, 20, 21, 22, 23, 24, or 25.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 8 to 28.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 15 to 38.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 15 to 28.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 8 to 25.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 15 to 25.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ta), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 10 to 38.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 10 to 25.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 12 to 38.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 12 to 25.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 14 to 38.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is an integer from 14 to 25.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, or 38.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, or 28.

In some embodiments, each Z of the modified antisense oligomers of thedisclosure, including compounds of formulas (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25.

In some embodiments, each Nu of the antisense oligomers of thedisclosure, including compounds of formula (I), (Ia), (IVa), (IVb),(IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and(VIII), is independently selected from the group consisting of adenine,guanine, thymine, uracil, cytosine, hypoxanthine, 2,6-diaminopurine,5-methyl cytosine, C5-propynyl-modified pyrimidines, and9-(aminoethoxy)phenoxazine.

In some embodiments, the targeting sequence of the antisense oligomersof the disclosure, including compounds of formula (I), (Ia), (IVa),(IVb), (IVc), (V), (VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe),and (VIII), is complementary to 10 or more contiguous nucleotides in atarget region within intron 1, intron 2, or exon 2 of a pre-mRNA of thehuman acid alpha-glucosidase (GAA) gene. In certain embodiments, thetargeting sequence of the antisense oligomers of the disclosure,including compounds of formula (I), (Ia), (IVa), (IVb), (IVc), (V),(VI), (VII), (VIIa), (VIIb), (VIIc), (VIId), (VIIe), and (VIII), is afragment of at least 12 contiguous nucleotides, or is variant having atleast 90% sequence identity to a sequence (where X can be selected fromuracil (U) or thymine (T).

Additional antisense oligomers/chemistries that can be used inaccordance with the present disclosure include those described in thefollowing patents and patent publications, the contents of which areincorporated herein by reference: PCT Publication Nos. WO/2007/002390;WO/2010/120820; and WO/2010/148249; U.S. Pat. No. 7,838,657; and U.S.Application No. 2011/0269820.

The antisense oligonucleotides can be prepared by stepwise solid-phasesynthesis, employing methods known in the art and described herein andin the references cited herein.

iii. Preparation of PMO-X with Basic Nitrogen Internucleoside Linkers

Morpholino subunits, the modified intersubunit linkages, and oligomerscomprising the same can be prepared as described, for example, in U.S.Pat. Nos. 5,185,444, and 7,943,762, which are incorporated by referencein their entireties. The morpholino subunits can be prepared accordingto the following general Reaction Scheme I.

Referring to Reaction Scheme 1, wherein B represents a base pairingmoiety and PG represents a protecting group, the morpholino subunits maybe prepared from the corresponding ribonucleoside (1) as shown. Themorpholino subunit (2) may be optionally protected by reaction with asuitable protecting group precursor, for example trityl chloride. The 3′protecting group is generally removed during solid-state oligomersynthesis as described in more detail below. The base pairing moiety maybe suitably protected for sold phase oligomer synthesis. Suitableprotecting groups include benzoyl for adenine and cytosine, phenylacetylfor guanine, and pivaloyloxymethyl for hypoxanthine (1). Thepivaloyloxymethyl group can be introduced onto the N1 position of thehypoxanthine heterocyclic base. Although an unprotected hypoxanthinesubunit, may be employed, yields in activation reactions are farsuperior when the base is protected. Other suitable protecting groupsinclude those disclosed in co-pending U.S. application Ser. No.12/271,040, which is hereby incorporated by reference in its entirety.

Reaction of 3 with the activated phosphorous compound 4, results inmorpholino subunits having the desired linkage moiety 5. Compounds ofstructure 4 can be prepared using any number of methods known to thoseof skill in the art. For example, such compounds may be prepared byreaction of the corresponding amine and phosphorous oxychloride. In thisregard, the amine starting material can be prepared using any methodknown in the art, for example those methods described in the Examplesand in U.S. Pat. No. 7,943,762.

Compounds of structure 5 can be used in solid-phase automated oligomersynthesis for preparation of oligomers comprising the intersubunitlinkages. Such methods are well known in the art. Briefly, a compound ofstructure 5 may be modified at the 5′ end to contain a linker to a solidsupport. For example, compound 5 may be linked to a solid support by alinker comprising L¹¹ and L¹⁵.

The morpholino oligomers containing any number of modified linkages maybe prepared using methods described herein, methods known in the artand/or described by reference herein. Also described in the examples areglobal modifications of morpholino oligomers prepared as previouslydescribed (see e.g., PCT publication WO2008036127).

The term “protecting group” refers to chemical moieties that block someor all reactive moieties of a compound and prevent such moieties fromparticipating in chemical reactions until the protective group isremoved, for example, those moieties listed and described in T. W.Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd ed.John Wiley & Sons (1999). It may be advantageous, where differentprotecting groups are employed, that each (different) protective groupbe removable by a different means. Protective groups that are cleavedunder totally disparate reaction conditions allow differential removalof such protecting groups. For example, protective groups can be removedby acid, base, and hydrogenolysis. Groups such as trityl,dimethoxytrityl, acetal and tert-butyldimethylsilyl are acid labile andmay be used to protect carboxy and hydroxy reactive moieties in thepresence of amino groups protected with Cbz groups, which are removableby hydrogenolysis, and Fmoc groups, which are base labile. Carboxylicacid moieties may be blocked with base labile groups such as, withoutlimitation, methyl, or ethyl, and hydroxy reactive moieties may beblocked with base labile groups such as acetyl in the presence of aminesblocked with acid labile groups such as tert-butyl carbamate or withcarbamates that are both acid and base stable but hydrolyticallyremovable.

Carboxylic acid and hydroxyl reactive moieties may also be blocked withhydrolytically removable protective groups such as the benzyl group,while amine groups may be blocked with base labile groups such as Fmoc.A particularly useful amine protecting group for the synthesis ofcompounds of Formula (I) is the trifluoroacetamide. Carboxylic acidreactive moieties may be blocked with oxidatively-removable protectivegroups such as 2,4-dimethoxybenzyl, while co-existing amino groups maybe blocked with fluoride labile silyl carbamates.

Allyl blocking groups are useful in the presence of acid- andbase-protecting groups since the former are stable and can besubsequently removed by metal or pi-acid catalysts. For example, anallyl-blocked carboxylic acid can be deprotected with apalladium(0)-catalyzed reaction in the presence of acid labile t-butylcarbamate or base-labile acetate amine protecting groups. Yet anotherform of protecting group is a resin to which a compound or intermediatemay be attached. As long as the residue is attached to the resin, thatfunctional group is blocked and cannot react. Once released from theresin, the functional group is available to react.

Typical blocking/protecting groups are known in the art and include, butare not limited to the following moieties:

Unless otherwise noted, all chemicals were obtained fromSigma-Aldrich-Fluka. Benzoyl adenosine, benzoyl cytidine, andphenylacetyl guanosine were obtained from Carbosynth Limited, UK.

Synthesis of PMO, PMO+, PPMO, and PMO-X containing further linkagemodifications as described herein was done using methods known in theart and described in pending U.S. application Ser. Nos. 12/271,036 and12/271,040 and PCT publication number WO/2009/064471, which are herebyincorporated by reference in their entirety.

PMO with a 3′ trityl modification are synthesized essentially asdescribed in PCT publication number WO/2009/064471 with the exceptionthat the detritylation step is omitted.

III. Formulations

The compounds of the present disclosure may also be admixed,encapsulated, conjugated or otherwise associated with other molecules,molecule structures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the disclosure encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the disclosure, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

As used herein, the term “prodrug” indicates a therapeutic agent that isprepared in an inactive form that is converted to an active form (i.e.,drug) within the body or cells thereof by the action of endogenousenzymes or other chemicals and/or conditions. In particular, prodrugversions of the oligomers of the disclosure are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

As used herein, the term “pharmaceutically acceptable salts” refers tophysiologically and pharmaceutically acceptable salts of the compoundsof the disclosure: i.e., salts that retain the desired biologicalactivity of the parent compound and do not impart undesiredtoxicological effects thereto. For oligomers, examples ofpharmaceutically acceptable salts and their uses are further describedin U.S. Pat. No. 6,287,860, which is incorporated herein in itsentirety.

The present disclosure also includes pharmaceutical compositions andformulations which include the antisense compounds of the disclosure.The pharmaceutical compositions of the present disclosure may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic and to mucousmembranes including vaginal and rectal delivery), pulmonary, e.g., byinhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; or intracranial, e.g., intrathecal or intraventricular,administration. Oligomers with at least one 2′-O-methoxyethylmodification are believed to be particularly useful for oraladministration. Pharmaceutical compositions and formulations for topicaladministration may include transdermal patches, ointments, lotions,creams, gels, drops, suppositories, sprays, liquids and powders.Conventional pharmaceutical carriers, aqueous, powder or oily bases,thickeners and the like may be necessary or desirable. Coated condoms,gloves and the like may also be useful.

The pharmaceutical formulations of the present disclosure, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present disclosure may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present disclosure may also beformulated as suspensions in aqueous, non-aqueous or mixed media.Aqueous suspensions may further contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension may alsocontain stabilizers.

Pharmaceutical compositions of the present disclosure include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent disclosure may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogeneous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present disclosure.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present disclosure include liposomal formulations.As used herein, the term “liposome” means a vesicle composed ofamphiphilic lipids arranged in a spherical bilayer or bilayers.Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior thatcontains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentdisclosure may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In some embodiments, the present disclosure employs various penetrationenhancers to affect the efficient delivery of nucleic acids,particularly oligomers. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Formulations for topical administration include those in which theoligomers of the disclosure are in admixture with a topical deliveryagent such as lipids, liposomes, fatty acids, fatty acid esters,steroids, chelating agents and surfactants. Lipids and liposomes includeneutral (e.g., dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic(e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolanine DOTMA).

For topical or other administration, oligomers of the disclosure may beencapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligomers may becomplexed to lipids, in particular to cationic lipids. Fatty acids andesters, pharmaceutically acceptable salts thereof, and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety. Topical formulations are described in detail inU.S. patent application Ser. No. 09/315,298 filed on May 20, 1999, whichis incorporated herein by reference in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Oral formulations are thosein which oligomers of the disclosure are administered in conjunctionwith one or more penetration enhancers, surfactants and chelators.Surfactants include fatty acids and/or esters or salts thereof, bileacids and/or salts thereof. Bile acids/salts and fatty acids and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. In some embodiments, the presentdisclosure provides combinations of penetration enhancers, for example,fatty acids/salts in combination with bile acids/salts. An exemplarycombination is the sodium salt of lauric acid, capric acid and UDCA.Further penetration enhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. Oligomers of the disclosure may bedelivered orally, in granular form including sprayed dried particles, orcomplexed to form micro or nanoparticles. Oligomer complexing agents andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety. Oral formulations for oligomers andtheir preparation are described in detail in U.S. application Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which isincorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the disclosure provide pharmaceuticalcompositions containing one or more oligomeric compounds and one or moreother chemotherapeutic agents which function by a non-antisensemechanism. Examples of such chemotherapeutic agents include but are notlimited to cancer chemotherapeutic drugs such as daunorubicin,daunomycin, dactinomycin, doxorubicin, epirubicin, idarubicin,esorubicin, bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxyco-formycin,4-hvdroxvperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the disclosure, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligomer), sequentially (e.g., 5-FU and oligomer for a period of timefollowed by MTX and oligomer), or in combination with one or more othersuch chemotherapeutic agents (e.g., 5-FU, MTX and oligomer, or 5-FU,radiotherapy and oligomer). Anti-inflammatory drugs, including but notlimited to nonsteroidal anti-inflammatory drugs and corticosteroids, andantiviral drugs, including but not limited to ribivirin, vidarabine,acyclovir and ganciclovir, may also be combined in compositions of thedisclosure. Combinations of antisense compounds and other non-antisensedrugs are also within the scope of this disclosure. Two or more combinedcompounds may be used together or sequentially.

In another related embodiment, compositions of the disclosure maycontain one or more antisense compounds, particularly oligomers,targeted to a first nucleic acid and one or more additional antisensecompounds targeted to a second nucleic acid target. Alternatively,compositions of the disclosure may contain two or more antisensecompounds targeted to different regions of the same nucleic acid target.Numerous examples of antisense compounds are known in the art. Two ormore combined compounds may be used together or sequentially.

IV. Methods of Use

In further aspects, the antisense oligomer compounds described hereinare used to treat diseases. In embodiments, the diseases are associatedwith a particular exon position or location wherein targeting at theexon position or location with the antisense oligomer compounds resultsin an increase or decrease of a mRNA or protein transcribed ortranslated from the exon position or location. In embodiments, anincrease is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to a control, for example, acontrol cell/subject. In embodiments, a decrease is 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to a control, for example, a control cell/subject.

In further aspects, the antisense oligomer compounds described hereinare used to treat diseases associated with a particular exon position orlocation, wherein the particular exon position or location contains atleast one string of three or more identical contiguous nucleobases inthe target sequence, and wherein targeting at the exon position orlocation with the antisense oligomer compounds results in an increase ordecrease of a mRNA or protein transcribed or translated from the exonposition or location. In embodiments, an increase is 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to a control, for example, a control cell/subject. Inembodiments, a decrease is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control, forexample, a control cell/subject.

In further aspects, the antisense oligomer compounds described hereinare used to treat diseases associated with a particular exon position orlocation, wherein the particular exon position or location contains atleast one biological palindrome sequence in the target sequence, andwherein targeting at the exon position or location with the antisenseoligomer compounds results in an increase or decrease of a mRNA orprotein transcribed or translated from the exon position or location. Inembodiments, an increase is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control, forexample, a control cell/subject. In embodiments, a decrease is 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% relative to a control, for example, a control cell/subject.

In embodiments, the antisense oligomer compounds described herein areused to treat an exon target associated with Duchenne muscular dystrophy(DMD) as described generally in PCT Publication No.: WO 2006/000057. Inembodiments, the target sequence comprises exon 44 in the processing ofhuman dystrophin pre-processed mRNA. In embodiments, the targetingsequence comprises any one of SEQ ID NOs: 2-7. In embodiments, thetarget sequence comprises exons 45, 51 or 53 of human dystrophinpre-processed mRNA. In embodiments, the targeting sequence comprises anyone of SEQ ID NOs: 75-123. In embodiments, targeting at the describedexon position or location with the antisense oligomer compounds resultsin an increase or decrease of a mRNA or protein transcribed ortranslated from the exon position or location. In embodiments, anincrease is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%. 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, or 100% relative to a control, for example, acontrol cell/subject. In embodiments, a decrease is 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to a control, for example, a control cell/subject.

In embodiments, the antisense oligomer compounds described herein areused to treat an exon target associated with spinal muscular atrophy(SMA) as described generally in WO 2017/040271, the content of which isincorporated herein in its entirety. In embodiments, the target sequencecomprises a region adjacent to exon 7 in the processing of human SMN2pre-processed mRNA. In embodiments, the targeting sequence comprises anyone of SEQ ID NOs: 9-25. In embodiments, targeting at the described exonposition or location with the antisense oligomer compounds results in anincrease or decrease of a mRNA or protein transcribed or translated fromthe exon position or location. In embodiments, an increase is 5%, 6%,7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or100% relative to a control, for example, a control cell/subject. Inembodiments, a decrease is 5%, 6%. 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%.15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control, forexample, a control cell/subject.

In embodiments, the antisense oligomer compounds described herein areused to treat an exon target associated with glycogen storage diseasetype II (GSD-II) as described generally in PCT Patent Application No.PCT/US 17/28002]. In embodiments, the target sequence comprises a regionassociated with exon 2 of the human acid alpha-glucosidase pre-processedmRNA. In embodiments, the targeting sequence comprises any one of SEQ IDNOs: 26-71. In embodiments, targeting at the described exon position orlocation with the antisense oligomer compounds results in an increase ordecrease of a mRNA or protein transcribed or translated from the exonposition or location. In embodiments, an increase is 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to a control, for example, a control cell/subject. Inembodiments, a decrease is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%,

In various aspects and embodiments, the antisense oligomer compoundsdescribed herein include a deletion sequence and are used to treatdiseases. In embodiments, the diseases are associated with a particularexon position or location, wherein targeting at the exon position orlocation with the antisense oligomer compounds results in an increase ordecrease of a mRNA or protein transcribed or translated from the exonposition or location. In embodiments, an increase is 5%, 6%, 7%, 8%, 9%,10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%relative to a control, for example, a control cell/subject. Inembodiments, a decrease is 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to a control, forexample, a control cell/subject. The deletion sequence comprises any oneof SEQ ID NOs: 1-128, wherein at least one nucleobase in any one of SEQID NOs: 1-128 has been deleted. In various embodiments, the deletionsequence comprises CTCCAACATCAAGGAAGATGGCATTTCTAG (SEQ ID NO: 69;Eteplirsen); GTTGCCTCCGGTTCTGAAGGTGTTC (SEQ ID NO: 70; Golodirsen); orCAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 71; Casimersen). In variousembodiments, the deletion sequence comprises any one of SEQ ID NOs:69-71, wherein at least one nucleobase in any one of SEQ ID NOs: 69-71has been deleted. In embodiments, the at least one nucleobase that hasbeen deleted is internal to the sequence of any one of SEQ ID NOs:69-71.

Accordingly, a physician or clinician may consider applying knowledgeobtained in relevant pharmacogenomics studies in determining whether toadminister a therapeutic agent as well as tailoring the dosage and/ortherapeutic regimen of treatment with a therapeutic agent.

Effective delivery of the antisense oligomer to the target nucleic acidis one aspect of treatment. Routes of antisense oligomer deliveryinclude, but are not limited to, various systemic routes, including oraland parenteral routes, e.g., intravenous, subcutaneous, intraperitoneal,and intramuscular, as well as inhalation, transdermal and topicaldelivery. The appropriate route may be determined by one of skill in theart, as appropriate to the condition of the subject under treatment.Vascular or extravascular circulation, the blood or lymph system, andthe cerebrospinal fluid are some non-limiting sites where the RNA may beintroduced. Direct CNS delivery may be employed, for instance,intracerebral ventribular or intrathecal administration may be used asroutes of administration.

In particular embodiments, the antisense oligomer(s) are administered tothe subject by intramuscular injection (IM), i.e., they are administeredor delivered intramuscularly. Non-limiting examples of intramuscularinjection sites include the deltoid muscle of the arm, the vastuslateralis muscle of the leg, and the ventrogluteal muscles of the hips,and dorsogluteal muscles of the buttocks. In specific embodiments, aPMO, PMO-X, or PPMO is administered by IM.

In certain embodiments, the subject in need thereof as glycogenaccumulation in central nervous system tissues. Examples includeinstances where central nervous system pathology contributes torespiratory deficits in GSD-II (see, e.g., DeRuisseau et al., PNAS USA.106:9419-24, 2009). Accordingly, the antisense oligomers describedherein can be delivered to the nervous system of a subject by anyart-recognized method, e.g., where the subject has GSD-II withinvolvement of the CNS. For example, peripheral blood injection of theantisense oligomers of the disclosure can be used to deliver saidreagents to peripheral neurons via diffusive and/or active means.Alternatively, the antisense oligomers can be modified to promotecrossing of the blood-brain-barrier (BBB) to achieve delivery of saidreagents to neuronal cells of the central nervous system (CNS). Specificrecent advancements in antisense oligomer technology and deliverystrategies have broadened the scope of antisense oligomer usage forneuronal disorders (see, e.g., Forte, A., et al. 2005. Curr. DrugTargets 6:21-29; Jaeger, L. B., and W. A. Banks. 2005. Methods Mol. Med.106:237-251; Vinogradov, S. V., et al. 2004. Bioconjug. Chem. 5:50-60;the foregoing is incorporated herein in their entirety by reference).For example, the antisense oligomers of the disclosure can be generatedas peptide nucleic acid (PNA) compounds. PNA reagents have each beenidentified to cross the BBB (Jaeger, L. B., and W. A. Banks. 2005.Methods Mol. Med. 106:237-251). Treatment of a subject with, e.g., avasoactive agent, has also been described to promote transport acrossthe BBB (Id). Tethering of the antisense oligomers of the disclosure toagents that are actively transported across the BBB may also be used asa delivery mechanism. Administration of antisense agents together withcontrast agents such as iohexol (e.g., separately, concurrently, in thesame formulation) can also facilitate delivery across the BBB, asdescribed in PCT Publication No. WO/2013/086207, incorporated byreference in its entirety.

In certain embodiments, the antisense oligomers of the disclosure can bedelivered by transdermal methods (e.g., via incorporation of theantisense oligomers into, e.g., emulsions, with such antisense oligomersoptionally packaged into liposomes). Such transdermal andemulsion/liposome-mediated methods of delivery are described fordelivery of antisense oligomers in the art, e.g., in U.S. Pat. No.6,965,025, the contents of which are incorporated in their entirety byreference herein.

The antisense oligomers described herein may also be delivered via animplantable device. Design of such a device is an art-recognizedprocess, with, e.g., synthetic implant design described in, e.g., U.S.Pat. No. 6,969,400, the contents of which are incorporated in theirentirety by reference herein.

Antisense oligomers can be introduced into cells using art-recognizedtechniques (e.g., transfection, electroporation, fusion, liposomes,colloidal polymeric particles and viral and non-viral vectors as well asother means known in the art). The method of delivery selected willdepend at least on the oligomer chemistry, the cells to be treated andthe location of the cells and will be apparent to the skilled artisan.For instance, localization can be achieved by liposomes with specificmarkers on the surface to direct the liposome, direct injection intotissue containing target cells, specific receptor-mediated uptake, orthe like.

As known in the art, antisense oligomers may be delivered using, e.g.,methods involving liposome-mediated uptake, lipid conjugates,polylysine-mediated uptake, nanoparticle-mediated uptake, andreceptor-mediated endocytosis, as well as additional non-endocytic modesof delivery, such as microinjection, permeabilization (e.g.,streptolysin-O permeabilization, anionic peptide permeabilization),electroporation, and various non-invasive non-endocytic methods ofdelivery that are known in the art (refer to Dokka and Rojanasakul,Advanced Drug Delivery Reviews 44, 35-49, incorporated by reference inits entirety).

The antisense oligomers may be administered in any convenient vehicle orcarrier which is physiologically and/or pharmaceutically acceptable.Such a composition may include any of a variety of standardpharmaceutically acceptable carriers employed by those of ordinary skillin the art. Examples include, but are not limited to, saline, phosphatebuffered saline (PBS), water, aqueous ethanol, emulsions, such asoil/water emulsions or triglyceride emulsions, tablets and capsules. Thechoice of suitable physiologically acceptable carrier will varydependent upon the chosen mode of administration. “Pharmaceuticallyacceptable carrier” is intended to include any and all solvents,dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents, and the like, compatible withpharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The compounds (e.g., antisense oligomers) of the present disclosure maygenerally be utilized as the free acid or free base. Alternatively, thecompounds of this disclosure may be used in the form of acid or baseaddition salts. Acid addition salts of the free amino compounds of thepresent disclosure may be prepared by methods well known in the art, andmay be formed from organic and inorganic acids. Suitable organic acidsinclude maleic, fumaric, benzoic, ascorbic, succinic, methanesulfonic,acetic, trifluoroacetic, oxalic, propionic, tartaric, salicylic, citric,gluconic, lactic, mandelic, cinnamic, aspartic, stearic, palmitic,glycolic, glutamic, and benzenesulfonic acids.

Suitable inorganic acids include hydrochloric, hydrobromic, sulfuric,phosphoric, and nitric acids. Base addition salts included those saltsthat form with the carboxylate anion and include salts formed withorganic and inorganic cations such as those chosen from the alkali andalkaline earth metals (for example, lithium, sodium, potassium,magnesium, barium and calcium), as well as the ammonium ion andsubstituted derivatives thereof (for example, dibenzylammonium,benzylammonium, 2-hydroxyethylammonium, and the like). Thus, the term“pharmaceutically acceptable salt” is intended to encompass any and allacceptable salt forms.

In addition, prodrugs are also included within the context of thisdisclosure. Prodrugs are any covalently bonded carriers that release acompound in vivo when such prodrug is administered to a patient.Prodrugs are generally prepared by modifying functional groups in a waysuch that the modification is cleaved, either by routine manipulation orin vivo, yielding the parent compound. Prodrugs include, for example,compounds of this disclosure wherein hydroxy, amine or sulfhydryl groupsare bonded to any group that, when administered to a patient, cleaves toform the hydroxy, amine or sulfhydryl groups. Thus, representativeexamples of prodrugs include (but are not limited to) acetate, formateand benzoate derivatives of alcohol and amine functional groups of theantisense oligomers of the disclosure. Further, in the case of acarboxylic acid (—COOH), esters may be employed, such as methyl esters,ethyl esters, and the like.

In some instances, liposomes may be employed to facilitate uptake of theantisense oligomer into cells (see, e.g., Williams, S. A., Leukemia10(12):1980-1989, 1996; Lappalainen et al., Antiviral Res. 23:119, 1994;Uhlmann et al., antisense oligomers: a new therapeutic principle,Chemical Reviews, Volume 90, No. 4, 25 pages 544-584, 1990; Gregoriadis,G., Chapter 14, Liposomes, Drug Carriers in Biology and Medicine, pp.287-341, Academic Press, 1979). Hydrogels may also be used as vehiclesfor antisense oligomer administration, for example, as described in WO93/01286. Alternatively, the oligomers may be administered inmicrospheres or microparticles. (See, e.g., Wu, G. Y. and Wu, C. H., J.Biol. Chem. 262:4429-4432, 30 1987). Alternatively, the use ofgas-filled microbubbles complexed with the antisense oligomers canenhance delivery to target tissues, as described in U.S. Pat. No.6,245,747. Sustained release compositions may also be used. These mayinclude semipermeable polymeric matrices in the form of shaped articlessuch as films or microcapsules.

In one embodiment, the antisense oligomer is administered to a mammaliansubject, e.g., human or domestic animal, exhibiting the symptoms of alysosomal storage disorder, in a suitable pharmaceutical carrier. In oneaspect of the method, the subject is a human subject, e.g., a patientdiagnosed as having GSD-II (Pompe disease). In one preferred embodiment,the antisense oligomer is contained in a pharmaceutically acceptablecarrier, and is delivered orally. In another preferred embodiment, theoligomer is contained in a pharmaceutically acceptable carrier, and isdelivered intravenously (i.v.).

In one embodiment, the antisense compound is administered in an amountand manner effective to result in a peak blood concentration of at least200-400 nM antisense oligomer. Typically, one or more doses of antisenseoligomer are administered, generally at regular intervals, for a periodof about one to two weeks. Preferred doses for oral administration arefrom about 1-1000 mg oligomer per 70 kg. In some cases, doses of greaterthan 1000 mg oligomer/patient may be necessary. For i.v. administration,preferred doses are from about 0.5 mg to 1000 mg oligomer per 70 kg. Theantisense oligomer may be administered at regular intervals for a shorttime period, e.g., daily for two weeks or less. However, in some casesthe oligomer is administered intermittently over a longer period oftime. Administration may be followed by, or concurrent with,administration of an antibiotic or other therapeutic treatment. Thetreatment regimen may be adjusted (dose, frequency, route, etc.) asindicated, based on the results of immunoassays, other biochemical testsand physiological examination of the subject under treatment.

An effective in vivo treatment regimen using the antisense oligomers ofthe disclosure may vary according to the duration, dose, frequency androute of administration, as well as the condition of the subject undertreatment (i.e., prophylactic administration versus administration inresponse to localized or systemic infection). Accordingly, such in vivotherapy will often require monitoring by tests appropriate to theparticular type of disorder under treatment, and correspondingadjustments in the dose or treatment regimen, in order to achieve anoptimal therapeutic outcome.

Treatment may be monitored, e.g., by general indicators of disease knownin the art. The efficacy of an in vivo administered antisense oligomerof the disclosure may be determined from biological samples (tissue,blood, urine etc.) taken from a subject prior to, during and subsequentto administration of the antisense oligomer. Assays of such samplesinclude (1) monitoring the presence or absence of heteroduplex formationwith target and non-target sequences, using procedures known to thoseskilled in the art, e.g., an electrophoretic gel mobility assay; (2)monitoring the amount of a mutant mRNA in relation to a reference normalmRNA or protein as determined by standard techniques such as RT-PCR,Northern blotting, ELISA or Western blotting.

In some embodiments, the antisense oligomer is actively taken up bymammalian cells. In further embodiments, the antisense oligomer may beconjugated to a transport moiety (e.g., transport peptide or CPP) asdescribed herein to facilitate such uptake.

V. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is affected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligomers, and cangenerally be estimated based on EC50s found to be effective in in vitroand in vivo animal models. In general, dosage is from 0.01 μg to 100 gper kg of body weight, and may be given once or more daily, weekly,monthly or yearly, or even once every 2 to 20 years. Persons of ordinaryskill in the art can easily estimate repetition rates for dosing basedon measured residence times and concentrations of the drug in bodilyfluids or tissues. Following successful treatment, it may be desirableto have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligomer is administered inmaintenance doses, ranging from 0.01 μg to 100 g per kg of body weight,once or more daily, to once every 20 years.

While the present disclosure has been described with specificity inaccordance with certain of its embodiments, the following examples serveonly to illustrate the disclosure and are not intended to limit thesame. Each of the references, patents, patent applications, GenBankaccession numbers, and the like recited in the present application areincorporated herein by reference in its entirety.

The following Examples illustrate various embodiments of the invention,without limitation.

EXAMPLES Example 1: Oligomers Targeting Sequences with Runs of MultipleNucleobases Form Aggregations

This Example details that sequences with multiple homogeneousnucleobases present a problem during PMO manufacturing.

Specifically, the formation of intramolecular G-quartets can result inaggregation in the synthesis of G-rich PMOs such as GAA PMOs. Further,sequences that contain a series of three or more homogeneous nucleobasessuch as adenine, thymine, or cytosine, present similar problems ofaggregation or other manufacturing issues and inefficiencies.

GAA PMOs, or other PMOs that have homogeneous strings of nucleobases,when analyzed by analytical strong cation exchange (SCX) HPLC result inseveral different peaks with quite different retention times. Themultiple peaks should correspond to different aggregate forms of theconjugate.

Evidence for aggregation comes from HPLC performed using a Thermo PropacSCX-20 column. The buffer system and the HPLC operating conditions areshown in Table 1. The following procedure is used to prepare a drugproduct test sample for a lyophilized drug product at 100 mg per vial.After warming the sample to room temperature, a 3 mL syringe and needleare used to dispense 2.1 mL WFI into a vial. The sample is vortexed sothat it dissolves completely. Prior to use, the sample is incubated atroom temperature. A 3 mL syringe and needle are used to aspirate 2.0 mLof air into the vial. The vial is inverted, and 2.0 mL of solution istransferred from the vial to a glass container. Using a volumetricpipette, 1 mL of sample solution is dispensed and transferred into a 50mL volumetric flask. The flask is filled to volume with PBS and mixed.

TABLE 1 Buffer System and HPLC Operating Conditions Mobile Phase A: 24mM H₃PO₄, 25% can Mobile Phase B: 24 mM H₃PO₄, 1.0M KCl, 25% can FlowRate: 1.0 mL/minute Column: Thermo ProPac SCX-20, 4 × 250 mm ColumnTemperature: 60° C. Autosampler Temperature:  4° C. Injector Volume: 10μL Detector Wavelength: 260 nm Needle Wash: 50% ACN in water Run Time:30 minutes Time % Mobile % Mobile Gradient Program: (Minutes) Phase APhase B 2.0 70 30 22.0 35 65 30.0 0 100 30.1 0 100 40.0 70 30

Expected chromatographic results are that the sequences with three ormore consecutive G's produce an aggregation peak that elute much laterthan the un-aggregated species. The later-eluting peaks correspond tomultimeric aggregate forms of the conjugate.

Further, it is possible that consecutive homogeneous nucleobase stringspresent a problem during PMO manufacturing. As the number of homogeneousnucleobases increases, it becomes more likely that unwanted N-1deletions will happen. For example, a sequence containing a string offour thymines could produce unwanted N-1 deletions such that the finalcompound will only have three thymines. Under these circumstances, thethree thymine compounds would then be considered to be an impurityMethods such as mass spectrometry can be used to decipher the N-1deletion impurities.

Example 2: Oligomers Containing Deletions that Target Sequences withRuns of Multiple Nucleobases Prevent Aggregations

To mediate the problems presented by strings of homogeneous nucleobasesthat impact PMO manufacturing, removal of a single base or multiplebases from the string of homogeneous nucleobases should correct theaggregation or other manufacturing problems. Table 2 shows the sequencesthat are fully complementary and the corresponding deletion sequencesthat will be tested that will resolve the issue presented in Example 1.In order to disrupt the aggregation of PMO or mediate the manufacturingproblems, PMOs will be synthesized with deletions for guanine andthymine.

TABLE 2 Representative deletion sequence compounds with deletions SEQCompound¹ Sequence² ID NO GAA-IVS1.A (−65−41)GGC GGC ACN CAC GGG GCN CNC AAA G 121 GAA-IVS1.A (−65−41)-GGGC GGC ACN CAC GGG CNC NCA AAG  62 GAA-IVS1.A (−65−41)-2GGGC GGC ACN CAC GG CNC NCA AAG 122 GAA-IVS1.A (−65−41)-3GGGC GGC ACN CAC GCN CNC AAA G  63 GAA-IVS1.A (−65−41)-4GGGC GGC ACN CAC CNC NCA AAG  64 GAA-IVS1.A (−76−52)CGG GGC NCN CAA AGC AGC NCN GAG A 123 GAA-IVS1.A (−76−52)-GCGG GCN CNC AAA GCA GCN CNG AGA  59 GAA-IVS1.A (−76−52)-2GCGG CN CNC AAA GCA GCN CNG AGA  34 GAA-IVS1.A (−76−52)-3GCGC NCN CAA AGC AGC NCN GAG A  60 GAA-IVS1.A (−76−52)-4GCCN CNC AAA GCA GCN CNG AGA  61 GAA-IVS1.A.(−180,−156)NGG GGA GAG GGC CAG AAG GAA GGG C 124 GAA-IVS1.A.(−180,−156)-GNGG GGA GAG GGC CAG AAG GAA GGC  52 GAA-IVS1.A.(−180,−156)-2GNGG GGA GAG GGC CAG AAG GAA GC  53 GAA-IVS1.A.(−180,−156)-3GNGG GGA GAG GGC CAG AAG GAA C  54 GAA-IVS1.A.(−189,−165)GGC CAG AAG GAA GGG CGA GAA AAG C 125 GAA-IVS1.A.(−189,−165)-GGGC CAG AAG GAA GGC GAG AAA AGC 126 GAA-IVS1.A.(−189,−165)-2GGGC CAG AAG GAA GCG AGA AAA GC 127 GAA-IVS1.A.(−189,−165)-3GGGC CAG AAG GAA CGA GAA AAG C 128 ¹Reference to “A” in a Compound suchas in GAA-IVS1A (0180,−156) et seq is reference to an acceptor splicesite. ²Reference to “N” in a Sequence includes the independent selectionof thymine (T) or uracil (U).

Analysis is performed under highly aggregating SCX HPLC conditions ormass spectrometry as specified in Example 1.

The deletion of guanines or thymines based on Table 2 sequences shouldhave an effect on the relative amount of aggregation or efficiency ofPMO manufacturing. With the deletion of just one or two nucleobases, thepercent conjugate in the aggregate form should decrease from that of thefully complementary form or the percentage of product that is thedesired structure should increase compared to the fully complementarystructure.

Based on the unexpected results from other Examples described hereinwhich demonstrate that internal deletions of nucleobases do notsignificantly decrease PMO activity (exon skipping or inclusion), it isrationally expected that deletion of one or two nucleobases to decreasethe number of homogeneous nucleobases in Table 2 will not impact theirfunctional activity.

Example 3: Exon Skipping Percentages for Certain Deletion SequenceCompounds that Target Exon 44

Deletion sequence oligomers were prepared having the sequences shown inTable 3.

TABLE 3 Deletion sequence oligomers targeting exon 44 SEQ Compound¹Sequence² ID NO H44A (−01+24) Fully NNC NCA ACA GAN CNG 1 complementaryNCA AAN CGC C H44A (−1+24) bleb1 NNC NCA ACG ANC NGN 2 CAA ANC GCCH44A (−1+24) bleb3 NNC NCA ACA GNC NGN 3 CAA ANC GCC H44A (−1+24) bleb4NNC NCA ACA GAC NGN 4 CAA ANC GCC H44A (−1+24) bleb2 NNC NCA ACA ANC NGN5 CAA ANC GCC H44A (−1+24) bleb6 NNC NCA ACA GAN CGN 6 CAA ANC GCCH44A (−1+24) bleb5 NNC NCA ACA GAN NGN 7 CAA ANC GCC ¹Reference to“A” in a Compound such as in H44A (−1+24) bleb1 et seq is reference toan acceptor splice site. ²Reference to “N” in a Sequence includes theindependent selection of thymine (T) or uracil (U).

Varying concentrations (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, and 0.625μM) of the compounds were tested and values of exon skipping % weredetermined according to PCI Patent Publication No. WO 2014/153220, thecontent of which is incorporated herein in its entirety. The results areshown in FIG. 1 . Additionally, as shown in FIG. 1 , the fold increasein potency over control for the compounds identified in Table 3 wasdetermined as shown in Table 4.

TABLE 4 Fold increase for deletion sequence oligomers targeting exon 44Fold increase in Compound¹ potency over control H44A (−01 + 24) Fullycomplementary 7.4 H44A (−1 + 24) bleb1 8.5 H44A (−1 + 24) bleb3 7.2 H44A(−1 + 24) bleb4 6.1 H44A (−1 + 24) bleb2 6.0 H44A (−1 + 24) bleb6 5.9H44A (−1 + 24) bleb5 3.3 ¹Reference to “A”in a Compound such as in H44A(−1 + 24) bleb1 et seq is reference to an acceptor splice site.

Example 4: Exon Inclusion Percentages for Certain Deletion SequenceCompounds that Target Exon 7

Deletion sequence oligomers were prepared having the sequences shown inTable 5 and FIG. 2 .

TABLE 5 Deletion sequence oligomers targeting exon 7 SEQ ID Compound¹Sequence^(2,3) NO SMN2.7D 10C-10-34 3′-GGNCGNAANACNN  8 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer1 3′-GGNCGNAANACNN  9 NCACNNAGAAXG-5′SMN2.7D(−10−34) blebmer2 3′-GGNCGNAANACNN 10 NCACNNAGAXNG-5′SMN2.7D(−10−34) blebmer3 3′-GGNCGNAANACNN 11 NCACNNAXAANG-5′SMN2.7D(−10−34) blebmer4 3′-GGNCGNAANACNN 12 NCACNNXGAANG-5′SMN2.7D(−10−34) blebmer5 3′-GGNCGNAANACNN 13 NCACNXAGAANG-5′SMN2.7D(−10−34) blebmer6 3′-GGNCGNAANACNN 14 NCAXNNAGAANG-5′SMN2.7D(−10−34) blebmer7 3′-GGNCGNAANACNN 15 NCXCNNAGAANG-5′SMN2.7D(−10−34) blebmer8 3′-GGNCGNAANACNN 16 NXACNNAGAANG-5′SMN2.7D(−10−34) blebmer9 3′-GGNCGNAANACNN 17 XCACNNAGAANG-5′SMN2.7D(−10−34) blebmer10 3′-GGNCGNAANAXNN 18 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer11 3′-GGNCGNAANXCNN 19 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer12 3′-GGNCGNAAXACNN 20 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer13 3′-GGNCGNAXNACNN 21 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer14 3′-GGNCGXAANACNN 22 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer15 3′-GGNCXNAANACNN 23 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer16 3′-GGNXGNAANACNN 24 NCACNNAGAANG-5′SMN2.7D(−10−34) blebmer17 3′-GGXCGNAANACNN 25 NCACNNAGAANG-5′ ¹Referenceto “D” in a Compound such as in SMN2.7D(−10−34) blebmer1 et seq isreference to a donor splice site. ²Reference to “N” in a Sequenceincludes the independent selection of thymine (T) or uracil (U).³Reference to “X” in a Sequence signifies a deletion at that nucleobaseposition.

Varying concentrations (1 μM and 0.1 μM) of the compounds were testedand values for exon 7 inclusion % were determined according to WO2017/040271, the content of which is incorporated herein in itsentirety. The results are shown in FIG. 3 . Gapmer/blebmer sequences 1,2, 3, 5, 8, 9, 11, and 14 showed the best activities.

Example 5: Exon Inclusion Percentages for Certain Deletion SequenceCompounds that Target Exon 7

Oligomers were prepared having the sequences shown in Table 5 herein.Varying concentrations (1 μM, 0.3 μM, 0.1 μM, 0.03 μM, and 0.01 μM) of asubset of the compounds were tested and values for exon 7 inclusion %were determined according to WO 2017/040271, the content of which isincorporated herein in its entirety. The results are shown in FIG. 4 .

Further, in a separate set of experiments, varying concentrations (1 μM,0.3 μM, 0.1 μM, 0.03 μM, and 0.01 μM) of a subset of the compounds weretested and values for exon 7 inclusion % were determined. The resultsare shown in FIG. 5 . Gapmer/blebmer sequences 2, 7, and 11 showed thebest activities in the data depicted in FIGS. 4 and 5 .

Example 6: Modulating GAA Enzyme Activity for Certain Deletion SequenceCompounds that Target Exon 2

Deletion sequences have been designed to treat Pompe disease (alsoreferred to herein as glycogen storage disease type LI). Detailed inTable 6 are representative oligomers designed for treating Pompedisease.

TABLE 6 Representative deletion sequences for treating Pompe diseaseTargeting Sequence (TS)² SEQ Coordinates¹ (5′-3′) ID NO GAA-IVS1.A.GGC CAG AAG GAA GGC GAG 26 (−189,−165)-G AAA AGC GAA-IVS1.A.GCC AGA AGG AAG GC GAG 27 (−190,−166)-G AAA AGC N GAA-IVS1.A.CCA GAA GGA AGG CGA GAA 28 (−191,−167)-G AAG CNC GAA-IVS1.A.CAG AAG GAA GGC GAG AAA 29 (−192,−168)-G AGC NCC GAA-IVS1.A.AGA AGG AAG GCG AGA AAA 30 (−193,−169)-G GCN CCA GAA-IVS1.A.GAA GGA AGG CGA GAA AAG 31 (−194,−170)-G CNC CAG GAA-IVS1.A.AAG GAA GGC GAG AAA AGC 32 (−195,−171)-G NCC AGC GAA-IVS1.A.AGG AAG GCG AGA AAA GCN 33 (−196,−172)-G CCA GCA GAA-IVS1.A.CGG CNC NCA AAG CAG CNC 34 (−76−52)-2G NGA GA GAA-IVS1.A.ACG GCN CNC AAA GCA GCN 35 (−75−51)-2G CNG AG GAA-IVS1.A.CAC GGC NCN CAA AGC AGC 36 (−74−50)-2G NCN GA GAA-IVS1.A.NCA CGG CNC NCA AAG CAG 37 (−73−49)-2G CNC NG GAA-IVS1.A.CNC ACG GCN CNC AAA GCA 38 (−72−48)-2G GCN CN GAA-IVS1.A.ACN CAC GGC NCN CAA AGC 39 (−71−47)-2G AGC NC GAA-IVS1.A.GCG GCA CNC ACG GCN CNC 40 (−66−42)-2G AAA GC GAA-IVS1.A.GGC GGC ACN CAC GGC NCN 41 (−65−41)-2G CAA AG GAA-IVS1.A.CGG CAC NCA CGG CNC NCA 42 (−67−43)-2G AAG CA GAA-IVS1.A.GCA CNC ACG GCN CNC AAA 43 (−69−45)-2G GCA GC GAA-IVS1.A.GGC ACN CAC GGC NCN CAA 44 (−68−44)-2G AGC AG GAA-IVS1.A.CAC NCA CGG CNC NCA AAG 45 (−70−46)-2G CAG CN GAA-IVS1.A.GCC AGA AGG AAG GCG AGA 46 (−189,−166)-G AAA GC GAA-IVS1.A.CCA GAA GGA AGG CGA GAA 47 (−189,−167)-G AAG C GAA-IVS1.A.CAG AAG GAA GGC GAG AAA 48 (−189,−168)-G AGC GAA-IVS1.A.GGC CAG AAG GAA GGC GAG 49 (−188,−165)-G AAA AG GAA-IVS1.A.GGC CAG AAG GAA GGC GAG 50 (−187,−165)-G AAA A GAA-IVS1.A.GGC CAG AAG GAA GGC GAG 51 (−186,−165)-G AAA GAA-IVS1.A.CGG CAC NCA CGGC NCN CAA 42 (−67−43)-2G AGC A GAA-IVS1.A.GCG GCA CNC ACGG CNC NCA 40 (−66−42)-2G AAG C GAA-IVS1.A.GGC GGC ACN CAC G GCN CNC 41 (−65−41)-2G AAA G GAA-IVS1.A.NGG GGA GAG GGC CAG AAG 52 (−180,−156)-G GAA GGC GAA-IVS1.A.NGG GGA GAG GGC CAG AAG 53 (−180,−156)-2G GAA GC GAA-IVS1.A.NGG GGA GAG GGC CAG AAG 54 (−180,−156)-3G GAA C GAA-IVS1.A.GGC CAG AAG GAA GCG AGA 55 (−189,−165)-2G AAA GC GAA-IVS1.A.GGC CAG AAG GAA CGA GAA 56 (−189,−165)-3G AAG C GAA-IVS1.A.AGG AAG CGA GAA AAG CNC 57 (−196,−172)-2G CAG CA GAA-IVS1.A.AGG AAG GAG AAA AGC NCC 58 (−196,−172)-3G AGC A GAA-IVS1.A.GGG GCN CNC AAA GCA GCN 59 (−76−52)-G CNG AGA GAA-IVS1.A.GGG NCN CAA AGC AGC NCN 60 (−76−52)-3G GAG A GAA-IVS1.A.CCN CNC AAA GCA GCN CNG 61 (−76−52)-4G AGA GAA-IVS1.A.GGC GGC ACN CAC GGG CNC 62 (−65−41)-G NCA AAG GAA-IVS1.A.GGC GGC ACN CAC GCN CNC 63 (−65−41)-3G AAA G GAA-IVS1.A.GGC GGC ACN CAC CNC NCA 64 (−65−41)-4G AAG GAA-IVS1.A.GCG GGA GGG GCG GCA CNC 65 (−57−33)-G ACG GGC GAA-IVS1.A.GCG GGA GGG GCG GCA CNC 66 (−57−33)-2G ACG GC GAA-IVS1.A.GCG GGA GGG GCG GCA CNC 67 (−57−33)-3G ACG C GAA-IVS1.A.GCG GGA GGG GCG GCA CNC 68 (−57−33)-4G ACC ¹Reference to “A” in aCompound such as in GAA-IVS1.A.(−75−51)-2G et seq is reference to anacceptor splice site. ²For any of the sequences detailed herein, each“N” is independently selected from thymine (T) or uracil (U).

Oligomers are prepared having the sequences shown in Table 6 herein.Varying concentrations of a subset of the compounds are tested andchanges in GAA enzyme activity are measured according to PCT ApplicationNo. PCT/US 17/28002, the content of which is incorporated herein in itsentirety.

Example 7: Exon Skipping Percentages for Certain Deletion SequenceCompounds that Target Exons 51, 53, and 45

Deletion sequences have been designed to targeting exons 51, 53, and 45of the dystrophin gene. Detailed in Table 7 are representative oligomersdesigned to targeting exons 51, 53, and 45.

TABLE 7 Representative deletion sequences fortargeting exons 51, 53, and 45 of the dystrophin gene SEQTargeting Sequence (TS)² ID Coordinates¹ (5′-3′) NO H51A(+66+95)D1CCCAACANCAAGGAAGANGGCANNNCNAG  72 H51A(+66+95)D2CNCAACANCAAGGAAGANGGCANNNCNAG  73 H51A(+66+95)D3CNCCACANCAAGGAAGANGGCANNNCNAG  74 H51A(+66+95)D4CNCCAAANCAAGGAAGANGGCANNNCNAG  75 H51A(+66+95)D5CNCCAACNCAAGGAAGANGGCANNNCNAG  76 H51A(+66+95)D6CNCCAACACAAGGAAGANGGCANNNCNAG  77 H51A(+66+95)D7CNCCAACANAAGGAAGANGGCANNNCNAG  78 H51A(+66+95)D8CNCCAACANCAGGAAGANGGCANNNCNAG  79 H51A(+66+95)D9CNCCAACANCAAGAAGANGGCANNNCNAG  80 H51A(+66+95)D10CNCCAACANCAAGGAGANGGCANNNCNAG  81 H51A(+66+95)D11CNCCAACANCAAGGAAANGGCANNNCNAG  82 H51A(+66+95)D12CNCCAACANCAAGGAAGNGGCANNNCNAG  83 H51A(+66+95)D13CNCCAACANCAAGGAAGAGGCANNNCNAG  84 H51A(+66+95)D14CNCCAACANCAAGGAAGANGCANNNCNAG  85 H51A(+66+95)D15CNCCAACANCAAGGAAGANGGANNNCNAG  86 H51A(+66+95)D16CNCCAACANCAAGGAAGANGGCNNNCNAG  87 H51A(+66+95)D17CNCCAACANCAAGGAAGANGGCANNCNAG  88 H51A(+66+95)D18CNCCAACANCAAGGAAGANGGCANNNNAG  89 H51A(+66+95)D19CNCCAACANCAAGGAAGANGGCANNNCAG  90 H51A(+66+95)D20CNCCAACANCAAGGAAGANGGCANNNCNG  91 H53A(+36+60)D1GNGCCNCCGGNNCNGAAGGNGNNC  92 H53A(+36+60)D2 GNNCCNCCGGNNCNGAAGGNGNNC  93H53A(+36+60)D3 GNNGCNCCGGNNCNGAAGGNGNNC  94 H53A(+36+60)D4GNNGCCCCGGNNCNGAAGGNGNNC  95 H53A(+36+60)D5 GNNGCCNCGGNNCNGAAGGNGNNC  96H53A(+36+60)D6 GNNGCCNCCGNNCNGAAGGNGNNC  97 H53A(+36+60)D7GNNGCCNCCGGNCNGAAGGNGNNC  98 H53A(+36+60)D8 GNNGCCNCCGGNNNGAAGGNGNNC  99H53A(+36+60)D9 GNNGCCNCCGGNNCGAAGGNGTNNC 100 H53A(+36+60)D10GNNGCCNCCGGNNCNAAGGNGNNC 101 H53A(+36+60)D11 GNNGCCNCCGGNNCNGAGGNGNNC102 H53A(+36+60)D12 GNNGCCNCCGGNNCNGAAGNGNNC 103 H53A(+36+60)D13GNNGCCNCCGGNNCNGAAGGGNNC 104 H53A(+36+60)D14 GNNGCCNCCGGNNCNGAAGGNNNC105 H53A(+36+60)D15 GNNGCCNCCGGNCNGAAGGNGNC 106 H45A(−03+16)D1CANGCCANCCNGGAGNNCCNG 107 H45A(−03+16)D2 CAAGCCANCCNGGAGNNCCNG 108H45A(−03+16)D3 CAANCCANCCNGGAGNNCCNG 109 H45A(−03+16)D4CAANGCANCCNGGAGNNCCNG 110 H45A(−03+16)D5 CAANGCCNCCNGGAGNNCCNG 111H45A(−03+16)D6 CAANGCCACCNGGAGNNCCNG 112 H45A(−03+16)D7CAANGCCANCNGGAGNNCCNG 113 H45A(−03+16)D8 CAANGCCANCCGGAGNNCCNG 114H45A(−03+16)D9 CAANGCCANCCNGAGNNCCNG 115 H45A(−03+16)D10CAANGCCANCCNGGGNNCCNG 116 H45A(−03+16)D11 CAANGCCANCCNGGANNCCNG 117H45A(−03+16)D12 CAANGCCANCCNGGAGNCCNG 118 H45A(−03+16)D13CAANGCCANCCNGGAGNNCNG 119 H45A(−03+16)D14 CAANGCCANCCNGGAGNNCCG 120¹Reference to “A” in a Compound such as in H51A(+66+95)D1 et seq isreference to an acceptor splice site. ²Reference to “N” in a Sequenceincludes the independent selection of thymine (T) or uracil (U).

Oligomers are prepared having the sequences shown in Table 7 herein.Varying concentrations of the compounds were tested and values of exonskipping % are determined according to PCT Patent Publication No. WO2014/153220, the content of which is incorporated herein in itsentirety.

Example 8: Reduced Aggregation after Intral -G Deletion(s) in TargetingSequences

Removal of an internal -G nucleotide(s) from targeting sequences resultsin reduced aggregation. HPLC was performed on SEQ ID NO: 125 and SEQ IDNO: 126. These sequences are identical except that SEQ ID NO: 126 has aninternal G deletion relative to SEQ ID NO: 125. Both sequences weretested as part of the PMO and PPMO backbone structures. HPLC wasperformed according to the procedure described in Example 1 using thebuffer system and HPLC operating conditions described in Table 1.

As shown in FIG. 6B, there is separation between the SEQ ID NO: 125 PMOpeak (8.961 mAU) and the SEQ ID NO: 125 PPMO peak (12.683 mAU); however,higher molecular weight aggregates are present (see, peaks at 14.058,14.867, 16.579, and 17.674 mAUs). This is in contrast to SEQ ID NO: 126PPMO in which the higher molecular weight aggregates have been resolved(FIG. 7B). No higher molecular weight aggregates are present in eitherthe SEQ ID NO: 125 PMO or the SEQ ID NO: 126 PMO (FIGS. 6A and 7A)

1. A modified antisense oligonucleotide or a pharmaceutically acceptablesalt thereof comprising a nucleic acid sequence consisting of any one ofSEQ ID NOs. 2-7, wherein the modified antisense oligonucleotide containsone or more morpholino groups. 2-4. (canceled)
 5. The modified antisenseoligonucleotide or pharmaceutically acceptable salt thereof of claim 1,wherein the modified antisense oligonucleotide is conjugated to apeptide. 6-18. (canceled)
 19. The modified antisense oligonucleotide orpharmaceutically acceptable salt thereof of claim 1, wherein themodified antisense oligonucleotide or pharmaceutically acceptable saltthereof promotes skipping of exon 44 in the processing of a humandystrophin pre-processed mRNA.
 20. A pharmaceutical compositioncomprising the modified antisense oligonucleotide or pharmaceuticallyacceptable salt thereof of claim 1, and a pharmaceutically acceptablecarrier.
 21. A method for treating a muscular dystrophy in a patient inneed thereof comprising administering to the patient the modifiedantisense oligonucleotide or pharmaceutically acceptable salt thereof ofclaim 1.